Non-human animals expressing pH-sensitive immunoglobulin sequences

ABSTRACT

Genetically modified non-human animals are provided that express an immunoglobulin variable domain that comprises at least one histidine, wherein the at least one histidine is encoded by a substitution of a non-histidine codon in the germline of the animal with a histidine codon, or the insertion of a histidine codon in a germline immunoglobulin nucleic acid sequence. Immunoglobulin genes comprising histidines in one or more CDRs, in an N-terminal region, and/or in a loop  4  region are also provided. Immunoglobulin variable domains comprising one or more histidines (e.g., histidine clusters) substituted for non-antigen-binding non-histidine residues. Non-human animals that are progeny of animals comprising modified heavy chain variable loci (V, D, J segments), modified light chain variable loci (V, J segments), and rearranged germline light chain genes (VJ sequences) are also provided. Non-human animals that make immunoglobulin domains that bind antigens in a pH-sensitive manner are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.13/834,129, filed on Mar. 15, 2013 and published as U.S. Publication No.2013/0247236 on Sep. 19, 2013, which claims the benefit of priority toU.S. Provisional Application No. 61/611,950, filed 16 Mar. 2012, U.S.Provisional Application No. 61/613,352, filed Mar. 20, 2012, U.S.Provisional Application No. 61/736,930, filed Dec. 13, 2012, and U.S.Provisional Application 61/612,126, filed Mar. 16, 2012. The entirecontents of each of the applications are incorporated herein byreference.

FIELD OF THE INVENTION

A genetically modified non-human animal that expresses antibodiescapable of binding to an antigen in a pH dependent manner. Geneticallymodified non-human animals that comprise immunoglobulin loci that aremodified to contain at least one substitution or insertion of a codonencoding a protonatable amino acid. Genetically modified non-humananimals that comprise immunoglobulin loci that are modified to containat least one histidine substitution and/or at least one histidineinsertion in an immunoglobulin heavy chain V, D, or J gene segment, orlight chain V or J segment, or rearranged heavy chain VDJ region orrearranged light chain VJ region thereof. Genetically modified non-humananimals that express immunoglobulins that exhibit pH sensitivity inantigen binding. Genetically modified animals that comprise B cellpopulations that are enriched with respect to immunoglobulin variabledomains that comprise at least one histidine. Genetically modifiednon-human animals that comprise clusters of two or more histidinespresent as insertions and/or substitutions in an immunoglobulin heavychain V, D, and/or J gene segment, and or a light chain V and/or J genesegment, and/or rearranged heavy chain VDJ sequences or rearranged lightchain VJ sequences thereof.

Genetically modified immunoglobulin loci of non-human animals comprisingan unrearranged human heavy chain variable region nucleotide sequence,wherein the unrearranged human heavy chain variable region nucleotidesequence comprises an addition of least one histidine codon or asubstitution of at least one endogenous non-histidine codon with ahistidine codon. Non-human animals, including rodents, e.g., mice andrats, comprising a genetically modified immunoglobulin locus in theirgenome an unrearranged human heavy chain variable region nucleotidesequence, wherein the unrearranged human heavy chain variable regionnucleotide sequence comprises an addition of least one histidine codonor a substitution of at least one endogenous non-histidine codon with ahistidine codon. Genetically engineered non-human animals capable ofexpressing an antigen-binding protein that is characterized bypH-dependent antigen binding, improved recyclability, and/or enhancedserum half-life.

BACKGROUND

Immunoglobulin binding domains find therapeutic use in a wide variety offormats, including the traditional antibody format of a homodimericimmunoglobulin heavy chain associated with a cognate light chain. Manyof these formats, including the traditional format, exhibitpharmacokinetic features in vivo that are suboptimal, due to a widevariety of factors. In recent decades, disparate approaches have beentried to improve pharmacokinetics. These include, e.g., increasinghydrodynamic radius to reduce renal clearance by conjugation to polymers(e.g., PEG; reviewed in, e.g., Duncan, R. (2006) Polymer conjugates asanticancer nanomedicines, Nat. Rev. Cancer 6:688-701); sialylation ofN-glycans (reviewed in, e.g., Stork, R. et al. N-glycosylation as novelstrategy to improve pharmacokinetic properties of bispecificsingle-chain diabodies, J. Biol. Chem. 283(12):7804-7812); Fcmodifications for promoting neutral pH Fc-FcRn binding while promotingrelease at endosomal pH and association with serum albumin (see, e.g.,Chuang et al. (2002) Pharmaceutical Strategies Utilizing RecombinantSerum Albumin, Pharm. Res. 19(5):569-577). In appropriate applicationsand for appropriate formats, each of these approaches may offer somebenefits.

However, there remains a need in the art for improving therapeuticeffects and modalities for biopharmaceuticals, including but not limitedto manipulating immunoglobulin variable domain structures to engineervariable domains that exhibit pH-dependent binding. There is a need forvariable domains for use in antigen-binding proteins of a variety offormats, wherein the variable domains (or antigen-binding fragmentsthereof) confer upon the antigen-binding protein pH sensitivity withrespect to binding a target antigen or receptor. There is also a need inthe art for systems and methods for generating pH-dependentimmunoglobulin variable domains and antigen-binding fragments thereof.There is a need for biological systems that can generate a widediversity of immunoglobulin variable domains, wherein the wide diversityis enriched with respect to titratable amino acids that may confer uponthe variable domain pH sensitivity, e.g., the ability to bind a targetantigen or epitope at one pH (e.g., a neutral, or high pH), yet releasethe target antigen or epitope at a second pH (e.g., a low, or endosomal,pH).

Immunoglobulin light chains in certain formats present uniquechallenges. Antibodies typically comprise a homodimeric heavy chaincomponent, wherein each heavy chain monomer is associated with anidentical light chain. Antibodies having a heterodimeric heavy chaincomponent (e.g., bispecific antibodies) are desirable as therapeuticantibodies. But making bispecific antibodies having a suitable lightchain component that can satisfactorily associate with each of the heavychains of a bispecific antibody has proved problematic.

In one approach, a light chain might be selected by surveying usagestatistics for all light chain variable domains, identifying the mostfrequently employed light chain in human antibodies, and pairing thatlight chain in vitro with the two heavy chains of differing specificity.

In another approach, a light chain might be selected by observing lightchain sequences in a phage display library (e.g., a phage displaylibrary comprising human light chain variable region sequences, e.g., ahuman scFv library) and selecting the most commonly used light chainvariable region from the library. The light chain can then be tested onthe two different heavy chains of interest.

In another approach, a light chain might be selected by assaying a phagedisplay library of light chain variable sequences using the heavy chainvariable sequences of both heavy chains of interest as probes. A lightchain that associates with both heavy chain variable sequences might beselected as a light chain for the heavy chains.

In another approach, a candidate light chain might be aligned with theheavy chains' cognate light chains, and modifications are made in thelight chain to more closely match sequence characteristics common to thecognate light chains of both heavy chains. If the chances ofimmunogenicity need to be minimized, the modifications preferably resultin sequences that are present in known human light chain sequences, suchthat proteolytic processing is unlikely to generate a T cell epitopebased on parameters and methods known in the art for assessing thelikelihood of immunogenicity (i.e., in silico as well as wet assays).

All of such approaches rely on in vitro methods that subsume a number ofa priori restraints, e.g., sequence identity, ability to associate withspecific pre-selected heavy chains, etc. There is a need in the art forcompositions and methods that do not rely on manipulating in vitroconditions, but that instead employ more biologically sensibleapproaches to making human epitope-binding proteins that include acommon light chain.

In addition, therapeutic antibodies, e.g., bispecific therapeuticantibodies, have some limitations in that they often require high dosesto achieve desired efficacy. This is partly due to the fact thatantibody-antigen complexes are internalized into the endosome, and aretargeted for lysosomal degradation in a process called target-mediatedclearance. Thus, there is a need in the art for methods and compositionsthat lead to more efficient antibody recycling, e.g., bispecificantibody recycling, and prevent degradation of the antibody by promotingdissociation of antibody-antigen complexes in the endosomal compartmentwithout compromising the specificity and affinity of the antibody towardthe antigen.

Drugs administered into the body, including therapeutic monoclonalantibodies, can be affected via various elimination mechanisms,including glomerular filtration (e.g., into urine), secretion (e.g.,into the bile), and catabolism by cells. While small molecules arecleared from the body via renal filtration, the majority of secretedantibodies (e.g., IgG, which are too big to be filtered throughglomeruli) are primarily removed from the body via cell-mediatedcatabolism, e.g., fluid-phase endocytosis (phagocytosis) orreceptor-mediated endocytosis. For example, soluble molecules withseveral repeated epitopes are bound by a plurality of circulatingantibodies, and the resulting large antigen-antibody complexes arephagocytosed rapidly into cells for degradation. On the other hand, cellsurface target receptors, which are bound by antibodies (i.e.,receptor-antibody complexes), undergo target-mediated endocytosis in adose-dependent manner, which leads to formation of endosomes destinedfor lysosomal degradation inside cells. In some cases, the endocytosedreceptor-antibody complexes bind neonatal Fc receptors (FcRn) inside theendosomes in a pH-dependent manner and are routed back to the cellsurface for release into plasma or interstitial fluids upon exposure toa neutral extracellular pH (e.g., pH 7.0-7.4).

There is a need in the art for systems, e.g., non-human animals, cells,and genomic loci that generate antigen-binding proteins with titratableresidues, e.g., genetically modified loci that rearrange immunoglobulingene segments to generate heavy chain variable domains that respond tochanges in pH, e.g., that donate or accept protons and, e.g., whosebinding characteristics differ according to protonation state.

There is also a need in the art for methods and compositions that canfurther increase recycling efficiency of endocytosed antigen-bindingproteins by promoting dissociation of antigen-binding proteins fromreceptor-antigen-binding protein complexes or by increasing the affinityof antigen-binding proteins toward FcRn in an acidic endosomalcompartment without compromising the specificity and affinity of theantigen-binding protein toward an antigen of interest.

SUMMARY

Compositions and methods are provided for making genetically modifiedanimals that make immunoglobulin variable domains that comprise at leastone histidine residue encoded by a germline modification of thenon-human animal, wherein the germline modification comprises at leastone of the insertion of a histidine codon into a heavy chain V, D, or Jsegment, insertion of a histidine codon into a light chain V or Jsegment, insertion of a histidine codon into a rearranged light chain VJgene, substitution of a non-histidine codon with a histidine codon in aheavy chain V, D, or J segment, substitution of a non-histidine codonwith a histidine codon in a light chain V or J segment, substitution ofa non-histidine codon with a histidine codon in a rearranged light chainVJ sequence.

Compositions and methods are also provided for introducing clusters ofhistidine codons in germline immunoglobulin sequences of non-humananimals.

Compositions and methods are also provided for introducing histidineinsertions, or substitutions of non-histidine codons with histidinecodons, in N-terminal-encoding regions of immunoglobulin genes, loop4-encoding regions of immunoglobulin genes, CDR-encoding regions ofimmunoglobulin genes (e.g., rearranged V(D)J sequences or V, (D), J genesegments).

Compositions and methods for making non-human animal progeny thatcomprise insertions of histidine codons and/or substitutions ofnon-histidine codons with histidine codons in both immunoglobulin heavychain loci and in immunoglobulin light chain loci.

In one aspect, a genetically modified non-human animal comprising in itsgermline an immunoglobulin locus comprising a substitution or aninsertion in an immunoglobulin variable locus of at least onenon-histidine codon with a histidine codon. In one embodiment, thevariable locus (e.g., an unrearranged V(D)J segments locus) comprises atleast a portion of a human variable (V(D)J segments) locus.

In one embodiment, the genetically modified non-human animal comprisesin its germline a first variable locus (e.g., an unrearrangedimmunoglobulin heavy chain (V(D)J segments locus) and a second variablelocus (e.g., an unrearranged immunoglobulin light chain (V,J segmentslocus; or a rearranged immunoglobulin light chain VJ sequence).

In one embodiment, the non-human animal comprises a first and a secondvariable locus, wherein at least the first or the second variable locuscomprises an insertion of at least one histidine codon or a substitutionof at least one non-histidine codon with a histidine codon.

In one embodiment, both the first and the second variable locus eachcomprise a substitution or insertion of at least one non-histidine codonwith a histidine codon.

In one embodiment, the first variable locus comprises at least afunctional portion of an unrearranged heavy chain variable locus(unrearranged V, D, J segments).

In one embodiment, the unrearranged heavy chain variable locus comprisesat least a portion of a human locus (unrearranged V, D,J segments).

In one embodiment, the unrearranged heavy chain locus is a human locuscomprising unrearranged V segments, a synthetic D segment that comprisesa linker, and a human J segment. In one embodiment, the synthetic Dsegment comprises at least one histidine codon.

In one embodiment, the second variable locus comprises at least afunctional portion of an unrearranged light chain locus (unrearrangedV,J segments).

In one embodiment, the second variable locus comprises a rearrangedimmunoglobulin light chain variable gene sequence (rearranged VJsequence).

In one embodiment, the substitution of an non-histidine codon with ahistidine codon and/or the insertion of a histidine codon is in anucleic acid sequence that encodes a variable domain and the histidineis in a region selected from an N-terminal region of an immunoglobulinchain, a loop 4 region of an immunoglobulin chain, a CDR1 of a heavychain, a CDR2 of a heavy chain, a CDR3 of a heavy chain, a CDR1 of alight chain, a CDR2 of a light chain, a CDR3 of a light chain, and acombination thereof.

In one embodiment, at least one of the first variable locus or thesecond variable locus is operably linked to an endogenous non-humanconstant region nucleic acid sequence at an endogenous non-humanimmunoglobulin locus.

In one embodiment the first variable locus (unrearranged human V, D, Jsegments) is operably linked to an endogenous non-human immunoglobulinheavy chain constant region nucleic acid sequence.

In one embodiment, the first variable locus (unrearrangd human V, D, Jsegments) is operably linked to the endogenous non-human immunoglobulinheavy chain constant region nucleic acid sequence at an endogenousnon-human immunoglobulin locus.

In one embodiment, the second variable locus (unrearranged V, Jsegments) is operably linked to an endogenous non-human immunoglobulinlight chain constant region sequence.

In one embodiment, the endogenous non-human immunoglobulin light chainconstant region sequence is at an endogenous non-human immunoglobulinlocus.

In one embodiment, the variable region sequence comprises a cluster of2, 3, 4, or 5 histidines that are substitutions of non-histidine codonswith histidine codons and/or insertions of histidine codons.

In one embodiment, the unrearranged heavy chain locus comprises D genesegments that are inverted with respect to the direction of orientationof the heavy chain locus.

In one embodiment, the inverted D segments are in a hydrophilic readingframe.

In one aspect, a genetically modified non-human animal is provided,comprising at least a portion of a human unrearranged immunoglobulinheavy chain variable region nucleic acid sequence (unrearranged V, D, Jsegments) operably linked to a constant region gene sequence, whereinone or more of the V, D, and J gene segments comprise at least onesubstitution of a non-histidine codon for a histidine codon, or at leastone histidine codon insertion; at least a portion of a humanunrearranged immunoglobulin light chain variable region nucleic acidsequence (unrearranged V, J segments) operably linked to a constantregion gene sequence, wherein one or more of the V and J gene segmentscomprise at least one substitution of a non-histidine codon for ahistidine codon, or at least one histidine codon insertion; wherein thenon-human animal expresses an immunoglobulin heavy chain variable domainand/or an immunoglobulin light chain variable domain that comprises ahistidine derived from a histidine substitution or insertion in thegermline of the mouse.

In one embodiment, the non-human animal is a mammal. In one embodiment,the mammal is a rodent. In one embodiment, the rodent is selected fromthe group consisting of a mouse, a rat, and a hamster.

In one embodiment, the human unrearranged immunoglobulin heavy chainvariable region nucleic acid sequence is operably linked to a non-humanconstant region sequence.

In one embodiment, the non-human constant region nucleic acid sequenceoperably linked to the human unrearranged immunoglobulin heavy chainvariable region nucleic acid sequence is at an endogenous non-humanimmunoglobulin locus in the germline of the non-human animal.

In one embodiment, the non-human constant region nucleic acid sequenceoperably linked to the human unrearranged immunoglobulin light chainvariable region nucleic acid sequence is at an endogenous non-humanimmunoglobulin locus in the germline of the non-human animal.

In one aspect, a genetically modified non-human animal is provided,comprising at least a portion of a human unrearranged immunoglobulinheavy chain variable region nucleic acid sequence (unrearranged V, D, Jsegments) operably linked to a constant region gene sequence, whereinone or more of the unrearranged V, D, and J gene segments comprise atleast one substitution of a non-histidine codon for a histidine codon,or at least one histidine codon insertion; a human rearrangedimmunoglobulin light chain variable region nucleic acid sequence(rearranged VJ sequence) operably linked to a light chain constantregion gene sequence, wherein the rearranged VJ sequence comprises atleast one substitution of a non-histidine codon for a histidine codon,or at least one histidine codon insertion; wherein the non-human animalexpresses an immunoglobulin heavy chain variable domain and/or animmunoglobulin light chain variable domain that comprises a histidinederived from a histidine substitution or insertion in the germline ofthe mouse.

In one embodiment, the genetically modified non-human animal is amammal. In one embodiment, the mammal is a rodent. In one embodiment,the rodent is selected from the group consisting of a mouse, a rat, anda hamster.

In one embodiment, the human unrearranged immunoglobulin heavy chainvariable region nucleic acid sequence is operably linked to a non-humanconstant region sequence. In one embodiment, the non-human constantregion sequence operably linked to the human unrearranged immunoglobulinheavy chain variable region nucleic acid sequence is at an endogenousnon-human immunoglobulin locus in the germline of the non-human animal.In one embodiment, the non-human constant region sequence operablylinked to the human rearranged immunoglobulin light chain variableregion nucleic acid sequence is at an endogenous non-humanimmunoglobulin locus in the germline of the non-human animal.

In one aspect, a genetically modified non-human animal is provided,wherein the animal comprises a B cell population that is characterizedby an enhanced presence of histidine residues in immunoglobulin heavyand light chains of the B cell population as compared with a wild-typenon-human animal. In one embodiment, the enhancement is about 2-4 fold.In one embodiment, the enhancement is about 2-10 fold.

In one aspect, a genetically modified non-human animal is provided thatexpresses immunoglobulin light and heavy chains that comprise histidinesencoded by substitutions and/or insertions in germline immunoglobulinsequences of the non-human animal.

In one aspect, a method is provided for making a non-human animal thatmakes antibody variable domains with histidines encoded by germlinehistidine codons, comprising: modifying the non-human animal in itsgermline to comprise at least one substitution of histidine codon for anon-histidine codon, or insertion of a histidine codon, in anunrearranged immunoglobulin heavy chain variable (unrearranged V, D, Jsegments) locus; and, modifying the non-human animal in its germline tocomprise at least one substitution of a histidine codon for anon-histidine codon, or insertion of a histidine codon, in anunrearranged immunoglobulin light chain variable (unrearranged V, Jsegments) locus.

In one embodiment, the method comprises genetically modifying thegermline of the mouse to comprise at least a portion of a humanunrearranged immunoglobulin heavy chain variable (V, D, J segments)locus, and making the histidine substitution or insertion in theunrearranged immunoglobulin heavy chain variable (unrearranged V, D, Jsegments) human locus.

In one embodiment, the method comprises genetically modifying thegermline of the mouse to comprise at least a portion of a humanunrearranged immunoglobulin light chain (unrearranged V, J segments)locus, and making the histidine substitution or insertion in theunrearranged human immunoglobulin light chain locus.

In one embodiment of the method, the non-human animal is a rodent. Inone embodiment, the rodent is selected from a mouse, a rat, and ahamster.

In one aspect, a method is provided for making a non-human animal thatmakes antibody variable domains with histidines encoded by germlinehistidine codons, comprising: modifying the non-human animal to compriseat least one substitution of histidine codon for a non-histidine codon,or insertion of a histidine codon, in an unrearranged immunoglobulinheavy chain variable (unrearranged V, D, J segments) locus; and,modifying the non-human animal to comprise at least one substitution ofa histidine codon for a non-histidine codon, or insertion of a histidinecodon, in a rearranged immunoglobulin light chain variable sequence(rearranged VJ sequence) in the germline.

In one embodiment of the method, the non-human animal is a rodent. Inone embodiment, the rodent is selected from a mouse, a rat, and ahamster.

In various aspects and embodiments, the non-human animals aregenetically modified by genetically modifying pluripotent or totipotentcells (e.g., embryonic stem (ES) cells), and employing the geneticallymodified cells as donor cells with a host embryo in a surrogate motherto gestate an animal derived from the genetically modified donor cells.In various aspects and embodiments, the non-human animals aregenetically modified by any other method known in the art.

Methods and compositions for making antibody variable domains thatexhibit a pH-dependent antigen binding are provided. Modifiedantigen-binding proteins are provided, as well as compositions andmethods for making them, that bind target antigen with low affinity at alow (e.g., endosomal) pH and that bind the same target antigen with highaffinity at a higher (e.g., extracellular), or neutral, pH.

In one aspect, a method for making an antibody that exhibitspH-dependent binding is provided, comprising modifying a sequence of avariable domain of the antibody to add a histidine residue, or tosubstitute an existing residue for a histidine residue, to form ahistidine-modified variable domain. In one embodiment, the substitutionis of a residue that is not critical for binding antigen (e.g., at aneutral or extracellular pH).

In one embodiment, two, three, four, five, or six or more residues aresubstituted to histidines. In one embodiment, the two, three, four,five, or six or more residues substituted to histidines are in acluster. In one embodiment, the cluster comprises two or moreconsecutive histidine substitutions. In one embodiment, the clustercomprises two or more histidine substitutions separated by one or morenon-histidine residues. In one embodiment, the cluster is 2, 3, 4, 5, 6,7, 8, 9, or 10 residues in length, and all residues not critical forbinding antigen (e.g., at a neutral or extracellular pH) are modified tohistidine.

In one embodiment the variable domain is a light chain variable domain(e.g., κ or λ). In one embodiment, the variable domain is in a heavychain variable domain. In one embodiment, the sequence of a light chainvariable domain and a heavy chain variable domain are modified.

In one embodiment, the sequence of the variable domain is a CDRsequence. In one embodiment, the CDR sequence is a CDR sequence of aheavy chain. In one embodiment, the CDR sequence is a CDR sequence of alight chain. In one embodiment, the CDR sequence is a CDR sequence of aheavy chain and a CDR sequence of a light chain.

In one embodiment, the CDR sequence is a CDR3 sequence. In oneembodiment, the CDR sequence is a CDR2 sequence. In one embodiment, theCDR sequence is a CDR3 sequence.

In one embodiment, the CDR sequence is a CDR1, a CDR2, and/or a CDR3sequence of a light chain. In one embodiment, the CDR sequence is aCDR1, a CDR2, and/or a CDR3 sequence of a heavy chain.

In one embodiment, the sequence of the variable domain of the antibodyis a loop 4 sequence. In one embodiment, the loop 4 sequence is a heavychain loop 4 sequence. In one embodiment, the loop 4 sequence is a lightchain loop 4 sequence.

In one embodiment, the sequence of the variable domain of the antibodyis an N-terminal sequence. In one embodiment, the N-terminal sequence isa heavy chain N-terminal sequence. In one embodiment, the N-terminalsequence is a light chain N-terminal sequence.

In one embodiment, the sequence of the variable domain of the antibodyis selected from a CDR sequence of a heavy chain, a CDR sequence of alight chain, a loop 4 sequence of a heavy chain, a loop 4 sequence of alight chain, an N-terminal sequence of a heavy chain, an N-terminalsequence of a light chain, and a combination thereof.

In one embodiment, the variable domain is from a heavy chain, and thesequence of the variable domain comprises a first CDR sequence and asequence selected from an N-terminal sequence, a loop 4 sequence, asecond CDR sequence, a third CDR sequence, and a combination thereof. Ina specific embodiment, the first CDR sequence is a CDR3, and thesequence of the variable domain further comprises a sequence selectedfrom an N-terminal sequence, a loop 4 sequence, a CDR2 sequence, a CDR1sequence, and a combination thereof.

In one embodiment, the histidine-modified variable domain is from aheavy chain, and the histidine modification is in a loop 4 sequence anda sequence selected from a CDR1 or CDR2 or CDR3, an N-terminal sequence,and a combination thereof. In a specific embodiment, the histidinemodification is in a loop 4 sequence and a CDR3 sequence. In a specificembodiment, the histidine modification is in a loop 4 sequence and aCDR3 sequence and an N-terminal sequence. In a specific embodiment, thehistidine modification is in a loop 4 sequence and an N-terminalsequence.

In one aspect, a his-modified immunoglobulin variable domain asdescribed herein is provided, wherein the his-modified immunoglobulinvariable domain that does not bind an antigen of interest or that bindsthe antigen of interest at a first affinity at a pH of less than 6; andbinds the same antigen of interest at a second affinity at a pH of about7 or more. In one embodiment the first pH is less than 5.5, or less than5. In one embodiment the first pH is 5.75. In one embodiment the secondpH is about 7 or higher. In one embodiment, the second pH is anextracellular pH of a human. In one embodiment, the second pH is 7.2 to7.4. In a specific embodiment, the second pH is 7.2.

In one embodiment, the his-modified variable domain comprises one, two,three, four, five, or six or more histidine substitutions in a sequenceselected from a CDR, an N-terminal, a loop 4, and a combination thereof.In a specific embodiment, the his-modified variable domain comprises amodification in a CDR3. In one embodiment, the his-modified variabledomain comprises a modification selected from a modification of a CDR3in a heavy chain, a modification of a CDR3 in a light chain, and acombination thereof. In one embodiment, the his-modified variable domaincomprises at least one substitution in a CDR (e.g., CDR3) and at leastone substitution in a sequence selected from an N-terminal, a loop 4,and a combination thereof.

In one embodiment, the CDR is selected from the group consisting of aheavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chainCDR1, a light chain CDR2, a light chain CDR3, and a combination thereof.

In one embodiment, the at least one CDR comprises a light chain CDR3. Inone embodiment, the at least one CDR comprises a light chain CDR3 and aheavy chain CDR3.

In one embodiment, the his-modified immunoglobulin variable domain bindsan antigen of interest at a neutral or basic pH (e.g., pH 7-7.4) with aK_(D) of about 10⁻⁶ or less (e.g., 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹,10⁻¹²), wherein the his-modified immunoglobulin variable domaincomprises: a CDR1 wherein all non-antigen-binding amino acid residuesare substituted with histidine, or wherein the CDR1 comprises a clusterof histidine substitutions. In one embodiment the variable domain doesnot bind the antigen of interest, or binds the antigen of interest10²-10⁶-fold weaker at an acidic pH (e.g., pH 5-6, in one embodiment, pH6).

In one embodiment, the his-modified immunoglobulin variable domain bindsan antigen of interest at a neutral or basic pH (e.g., pH 7-7.4) with aK_(D) of about 10⁻⁶ or less (e.g., 10⁻⁷, 10⁻⁹, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹,10⁻¹²), wherein the his-modified immunoglobulin variable domaincomprises a CDR2 wherein all non-antigen-binding amino acid residues aresubstituted with histidine, or wherein the CDR2 comprises a cluster ofhistidine substitutions. In one embodiment the variable domain does notbind the antigen of interest, or binds the antigen of interest10²-10⁶-fold weaker at an acidic pH (e.g., pH 5-6, in one embodiment, pH6).

In one embodiment, the his-modified immunoglobulin variable domain bindsan antigen of interest at a neutral or basic pH (e.g., pH 7-7.4) with aK_(D) of about 10⁻⁶ or less (e.g., 10⁻⁷, 10⁻⁹, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹,10⁻¹²), wherein the his-modified immunoglobulin variable domaincomprises a CDR3 wherein all non-antigen-binding amino acid residues aresubstituted with histidine, or wherein the CDR3 comprises a cluster ofhistidine substitutions. In one embodiment the variable domain does notbind the antigen of interest, or binds the antigen of interest10²-10⁶-fold weaker at an acidic pH (e.g., pH 5-6, in one embodiment, pH6).

In one aspect, a method is provided for making a human antigen-bindingpolypeptide comprising a his-modified domain, the method comprisingmodifying an immunoglobulin variable domain nucleotide sequence asdescribed herein to encode one or more histidines to form a nucleic acidsequence encoding a his-modified domain, and fusing the nucleic acidsequence encoding the his-modified domain (directly or with a linker) toa human immunoglobulin sequence.

In one embodiment, the human immunoglobulin sequence is animmunoglobulin constant domain sequence. In a specific embodiment, thehuman immunoglobulin constant domain sequence encodes an amino acidsequence selected from the group consisting of a C_(H)1, a hinge, aC_(H)2, a C_(H)3, and a combination thereof.

In one aspect, a cell that expresses a his-modified variable domain isprovided, wherein the his-modified variable domain is modified asdescribed herein. In one embodiment, the cell is a mammalian cell. Inone embodiment, the cell is selected from a HeLa cell, a DU145 cell, aLncap cell, a MCF-7 cell, a MDA-MB-438 cell, a PC3 cell, a T47D cell, aTHP-1 cell, a U87 cell, a SHSY5Y (human neuroblastoma) cell, a Saos-2cell, a Vero cell, a CHO cell, a GH3 cell, a PC12 cell, a human retinalcell (e.g., a PERC.6™ cell) and a MC3T3 cell. In a specific embodiment,the cell is a CHO cell.

In one aspect, a his-modified immunoglobulin variable domain asdescribed herein is provided, wherein the his-modified immunoglobulinvariable domain does not bind an antigen of interest or binds theantigen of interest at a first affinity at a pH of 5-6 (e.g., 5.75) andbinds the same antigen of interest at a second affinity at a pH of 7-7.4(e.g., 7.2), wherein at least one CDR comprises two or more histidinesubstitutions, and at least one non-CDR sequence comprises one or morehistidine substitutions wherein the at least one non-CDR sequence isselected from an N-terminal sequence, a loop 4 sequence, and acombination thereof.

In one embodiment, the first affinity is characterized by no binding, ora K_(D) of 10⁻⁶ or higher (e.g., 10⁻³), and the second affinity ischaracterized as being at least 2-fold, at least 5-fold, at least10-fold, at least 10²-fold, at least 10³-fold, at least 10⁴-fold, atleast 10⁵-fold, or at least 10⁶-fold stronger than the first affinity.

In one embodiment, the non-CDR sequence is on the same polypeptide asthe at least one CDR sequence. In one embodiment, the non-CDR sequenceis on a different polypeptide as the at least one CDR sequence.

In one embodiment, the at least one CDR is a CDR 3 of a heavy and/orlight chain, and the CDR3 comprises a substitution of at least half ofthe non-antigen-binding amino acid residues to histidine. In a specificembodiment, all of the non-antigen-binding amino acid residues of theCDR3 are substituted to histidine.

In one embodiment, the at least one CDR is a CDR3 of a heavy and/orlight chain, and the CDR3 comprises a substitution of three or morenon-antigen-binding amino acid residues to histidine. In one embodiment,four or more of the non-antigen-binding amino acid residues aresubstituted to histidine.

In one embodiment, the at least one CDR is a CDR3 of a heavy and/orlight chain, and the CDR3 comprises a substitution of two or morecontiguous non-antigen-binding amino acid residues to histidine. In oneembodiment, the CDR3 comprises a substitution of three or morecontiguous non-antigen-binding amino acids residues to histidine.

In one embodiment, the at least one CDR is a CDR3 of a light and/or aheavy chain, and further comprises a CDR selected from a light chainCDR1, a light chain CDR2, and a combination thereof.

In one embodiment, the at least one CDR is a CDR3 of a light and/or aheavy chain, and further comprises a CDR selected from a heavy chainCDR1, a heavy chain CDR2, and a combination thereof.

In one embodiment, the CDR is selected from the group consisting of aheavy chain CDR1, a heavy chain CDR2, a heavy chain CDR3, a light chainCDR1, a light chain CDR2, a light chain CDR3, and a combination thereof.

In one embodiment, the at least one CDR comprises a light chain CDR3. Inone embodiment, the at least one CDR comprises a light chain CDR3 and aheavy chain CDR3.

In one embodiment, the at least one CDR is a CDR3 of light and/or aheavy chain, and the at least one non-CDR sequence is a loop 4 sequence,wherein the loop 4 sequence comprises one or more histidinesubstitutions.

In one embodiment, the at least one CDR is a CDR3 of light and/or aheavy chain, and the at least one non-CDR sequence is an N-terminalsequence, wherein the N-terminal sequence comprises one or morehistidine substitutions.

In one embodiment, the at least one CDR is a CDR3 of a light chain, theat least one non-CDR sequence comprises an N-terminal sequence with oneor more histidine substitutions and a loop 4 sequence with one or morehistidine substitutions.

In one embodiment, the at least one CDR is a CDR3 of a heavy chain, theat least one non-CDR sequence comprises an N-terminal sequence with oneor more histidine substitutions and a loop 4 sequence with one or morehistidine substitutions.

In one embodiment, the his-modified immunoglobulin variable domain bindsan antigen of interest at pH 7-7.4 (e.g., pH 7.2) with a K_(D) of about10⁻⁷ or less (e.g., 10⁻⁹, 10⁻⁹, 10⁻¹¹, 10⁻¹²), wherein the his-modifiedimmunoglobulin variable domain comprises a CDR1 wherein allnon-antigen-binding amino acid residues are substituted with histidine.

In one embodiment, the his-modified immunoglobulin variable domain bindsan antigen of interest at pH 7-7.4 (e.g., pH 7.2) with a K_(D) of about10⁻⁷ or less (e.g., 10⁻⁹, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹²), wherein thehis-modified immunoglobulin variable domain comprises a CDR2 wherein allnon-antigen-binding amino acid residues are substituted with histidine.

In one embodiment, the his-modified immunoglobulin variable domain bindsan antigen of interest at pH 7-7.4 (e.g., pH 7.2) with a K_(D) of about10⁻⁷ or less (e.g., 10⁻⁹, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹²), wherein thehis-modified immunoglobulin variable domain comprises a CDR3 wherein allnon-antigen-binding amino acid residues are substituted with histidine.

In one aspect, use of a method as described herein in the manufacture ofa medicament for treating a human disease or disorder is provided. Inone embodiment, the medicament is an antibody. In a specific embodiment,the antibody is a human antibody.

In one aspect, use of a his-modified variable domain as described hereinin the manufacture of a medicament for treating a human disease ordisorder is provided. In one embodiment, the medicament is an antibody.In a specific embodiment, the antibody is a human antibody.

In one aspect, use of a method or his-modified variable domain asdescribed herein in the manufacture of a medicament for treating a humandisease or disorder is provided, wherein the medicament comprises anantigen-binding protein selected from an antibody, a multi-specificantibody (e.g., a bi-specific antibody), an scFv, a bi-specific scFv, adiabody, a triabody, a tetrabody, a V-NAR, a VHH, a VL, a F(ab), aF(ab)₂, a DVD (i.e., dual variable domain antigen-binding protein), anSVD (i.e., single variable domain antigen-binding protein), or abispecific T-cell engager (i.e., a BiTE).

In one aspect, a method as described herein is employed to generate aheavy and a κ or a λ light chain variable region sequence for making ahuman antigen-binding protein, further comprising fusing heavy and/orlight chain his-modified variable region sequences (directly or througha linker) to human heavy and light chain constant region sequences toform fused sequences, expressing the fused sequences in a cell, andrecovering an expressed antigen-binding protein comprising the fusedsequences. In various embodiments, the human heavy chain constantregions are selected from IgM, IgD, IgA, IgE and IgG. In variousspecific embodiments, the IgG is selected from an IgG1, an IgG2, an IgG3and an IgG4. In various embodiments, the human heavy chain constantregion is selected from a sequence comprising a C_(H)1, a hinge, aC_(H)2, a C_(H)3, a C_(H)4, and a combination thereof. In a specificembodiment the combination is a C_(H)1, a hinge, a C_(H)2, and a C_(H)3.In a specific embodiment the combination is a C_(H)1, a C_(H)2, and aC_(H)3. In a specific embodiment the combination is a hinge, a C_(H)2,and a C_(H)3. In a specific embodiment the combination is a hinge, aC_(H)2, and a C_(H)3.

In one aspect, a biological system is provided for generating anantibody or an antibody variable domain that binds a target antigen at aneutral pH but exhibits reduced binding of the same antigen at an acidicpH (e.g., pH 5.0-6.0). The biological system comprises a non-humananimal, e.g., a rodent (e.g, a mouse or rat) that has a rearranged lightchain sequence (e.g., a rearranged V-J) that comprises one or morehistidine modifications. In various aspects, the one or more histidinemodifications are in the light chain CDR3 codon. In various aspects, thenon-human animal comprises a human or humanized heavy chainimmunoglobulin locus. In various aspects, the non-human animal comprisesa replacement of endogenous non-human heavy chain variable gene segmentswith one or more human heavy chain V_(H), D_(H), and J_(H) segments,wherein the human segments are operably linked to a non-humanimmunoglobulin constant region. In various aspects, non-human animalswith universal light chains comprising light chain variable domains withsubstitutions of non-histidine residues for histidine residues areprovided. In various aspects these histidine-modified universal lightchain non-human animals (e.g., rodents, e.g., mice) are referred to ashistidine-universal light chain mice, histidine-ULC mice, or HULC mice.

Thus, in one aspect, provided herein is a genetically modified non-humananimal that comprises in its germline an immunoglobulin light chainlocus that comprises a single rearranged human immunoglobulin lightchain variable region gene sequence comprising human V_(L) and J_(L)segment sequences, wherein the single rearranged human immunoglobulinlight chain variable region sequence comprises a substitution of atleast one non-histidine codon with a histidine codon. In one embodiment,the single rearranged human immunoglobulin variable region sequence isoperably linked to an immunoglobulin light chain constant region genesequence. In one embodiment, the immunoglobulin light chain constantregion gene sequence is a non-human immunoglobulin light chain constantregion gene sequence. In one embodiment, the non-human immunoglobulinlight chain constant region gene sequence is an endogenousimmunoglobulin light chain constant region gene sequence. In oneembodiment, the non-human animal lacks a functional unrearrangedimmunoglobulin light chain variable region. In one embodiment, theimmunoglobulin light chain locus is at an endogenous non-humanimmunoglobulin light chain locus.

In one embodiment, the animal further comprises in its germline animmunoglobulin heavy chain locus that comprises an unrearrangedimmunoglobulin heavy chain variable region gene sequence comprisinghuman V_(H), D_(H), and J_(H) segments operably linked to animmunoglobulin heavy chain constant region gene sequence. In oneembodiment, the immunoglobulin heavy chain constant region gene sequenceis a non-human heavy chain constant region gene sequence. In oneembodiment, the non-human heavy chain constant region gene sequence isan endogenous immunoglobulin heavy chain constant region gene sequence.In one embodiment, the immunoglobulin heavy chain locus is at anendogenous immunoglobulin heavy chain locus.

In one embodiment, the substitution of at least one non-histidine codonwith a histidine codon is in the nucleotide sequence encoding acomplementary determining region (CDR). In one embodiment, thesubstitution of at least one non-histidine codon with a histidine codonis in the nucleotide sequence encoding a CDR3. In one embodiment, thesubstitution is of one, two, three, four, or more CDR3 codons. In oneaspect, the single rearranged human immunoglobulin light chain variableregion sequence comprised at the immunoglobulin light chain locus isderived from a human Vκ1-39 or Vκ3-20 gene segment. In one embodiment,the single rearranged human immunoglobulin light chain variable regionis derived from a rearranged Vκ1-39/Jκ5 or Vκ3-20/Jκ1 gene sequence. Inone embodiment, the single rearranged human immunoglobulin light chainvariable region is derived from a rearranged Vκ1-39/Jκ5 gene sequence,and the Vκ1-39/Jκ5 gene sequence comprises a replacement of at least onenon-histidine codon with a histidine codon designed to express ahistidine at a position selected from 105, 106, 108, 111, and acombination thereof. In another embodiment, the single rearranged humanimmunoglobulin light chain variable region is derived from a rearrangedVκ3-20/Jκ1 gene sequence, and the Vκ3-20/Jκ1 gene sequence comprises areplacement of at least one non-histidine codon with a histidine codondesigned to express a histidine at a position selected from 105, 106,107, 109, and a combination thereof.

In one aspect, the non-human animal described herein comprises apopulation of B cells in response to an antigen of interest that isenriched for antibodies that exhibit a decrease in dissociativehalf-life (t_(1/2)) at an acidic pH as compared to neutral pH of atleast about 2-fold, at least about 3-fold, at least about 4-fold, atleast about 5-fold, at least about 10-fold, at least about 15-fold, atleast about 20-fold, at least about 25-fold, or at least about 30-fold.In one embodiment, the decrease in t_(1/2) at an acidic pH as comparedto a neutral pH is about 30 fold or more.

In one embodiment, the animal expresses an antibody comprising a humanimmunoglobulin light chain variable domain with a substitution of atleast one non-histidine residue with a histidine residue at an aminoacid position encoded by the at least one codon substituted in theimmunoglobulin light chain variable region gene sequence. In oneembodiment, the animal expresses an antibody that retains a substitutionof at least one non-histidine residue with a histidine residue in anexpressed human immunoglobulin light chain variable domain, despitesomatic hypermutations.

In one embodiment, the non-human animal is a mammal. In one embodiment,the mammal is a rodent, e.g., a rat or a mouse. In one embodiment, thenon-human animal is a mouse. Thus, in one aspect, also provided hereinis a genetically modified mouse comprising in its germline animmunoglobulin light chain locus that comprises a single rearrangedhuman immunoglobulin light chain variable region gene sequencecomprising human V_(L) and J_(L) segment sequences, wherein the singlerearranged human immunoglobulin light chain variable region sequencecomprises a substitution of at least one non-histidine codon with ahistidine. In one embodiment, the mouse lacks a functional unrearrangedimmunoglobulin light chain variable region.

In one embodiment, the single rearranged immunoglobulin light chainvariable region gene sequence in the germline of the mouse is operablylinked to an immunoglobulin light chain constant region gene sequence.In one embodiment, the immunoglobulin light chain constant region genesequence is selected from a rat or a mouse immunoglobulin light chainconstant region gene sequence. In one embodiment, the immunoglobulinlight chain constant region gene sequence is a mouse sequence. In oneembodiment, the immunoglobulin light chain locus is at an endogenousmouse immunoglobulin light chain locus.

In a further embodiment, the mouse also comprises in its germline animmunoglobulin heavy chain locus that comprises an unrearrangedimmunoglobulin heavy chain variable region sequence comprising humanV_(H), D_(H), and J_(H) segments operably linked to an immunoglobulinheavy chain constant region gene sequence. In one aspect, theimmunoglobulin heavy chain constant region gene sequence is a rat or amouse heavy chain constant region gene sequence. In one embodiment, theimmunoglobulin heavy chain constant region gene sequence is a mousesequence. In one embodiment, the immunoglobulin heavy chain locus is atan endogenous mouse immunoglobulin heavy chain locus.

In one aspect, the mouse comprises a substitution of at least onenon-histidine codon with a histidine codon wherein the substitution isin the nucleotide sequence encoding a CDR. In one embodiment, thesubstitution is in a CDR3 codon, e.g., in one, two, three, four, or moreCDR3 codons. In one embodiment, the immunoglobulin light chain locus ofthe mouse comprises the single rearranged human immunoglobulin lightchain variable region sequence derived from a human Vκ1-39 or Vκ3-20gene segment, e.g., the single rearranged immunoglobulin light chainvariable region sequence is derived from a rearranged Vκ1-39/Jκ5 orVκ3-20/Jκ1 gene sequence. In one embodiment, the single rearrangedimmunoglobulin light chain variable region sequence is derived from arearranged Vκ1-39/Jκ5 gene sequence and the Vκ1-39/Jκ5 sequencecomprises a replacement of at least one non-histidine codon with ahistidine codon designed to express a histidine at a position selectedfrom 105, 106, 108, 111, and a combination thereof. In one embodiment,such replacement is designed to replace histidines at positions 105,106, 108, and 111. In another embodiment, such replacement is designedto replace histidines at positions 106, 108, and 111.

In another embodiment, the single rearranged immunoglobulin light chainvariable region sequence is derived from a rearranged Vκ3-20/Jκ1 genesequence and the Vκ3-20/Jκ1 sequence comprises a replacement of at leastone non-histidine codon with a histidine codon designed to express ahistidine at a position selected from 105, 106, 107, 109, and acombination thereof. In one embodiment, such replacement is designed toreplace histidines at positions 105, 106, 107, and 109. In anotherembodiment, such replacement is designed to replace histidines atpositions 105, 106, and 109.

In one embodiment, the mouse described herein comprises a population ofB cells in response to an antigen of interest that is enriched forantibodies that exhibit a decrease in dissociative half-life (t_(1/2))at an acidic pH as compared to neutral pH of at least about 2-fold, atleast about 3-fold, at least about 4-fold, at least about 5-fold, atleast about 10-fold, at least about 15-fold, at least about 20-fold, atleast about 25-fold, or at least about 30-fold. In one embodiment, thedecrease in t_(1/2) at an acidic pH as compared to a neutral pH is about30 fold or more.

In one embodiment, the mouse described herein expresses a population ofantigen-specific antibodies in response to an antigen of interestwherein all antibodies comprise (a) immunoglobulin light chain variabledomains derived from the same single rearranged human light chainvariable region gene sequence which comprises a substitution of at leastone non-histidine codon with a histidine codon, and (b) immunoglobulinheavy chains comprising heavy chain variable domains derived from arepertoire of human heavy chain V, D, and J segments.

Also provided herein is a non-human locus, e.g., mouse locus, comprisinga single rearranged human immunoglobulin light chain variable regiongene sequence comprising human V_(L) and J_(L) segment sequences,wherein the single rearranged human immunoglobulin light chain variableregion gene sequence comprises a substitution of at least onenon-histidine codon with a histidine codon. In one embodiment, the locusis comprised in the germline of a non-human animal. In one embodiment,the locus comprises the single rearranged human immunoglobulin lightchain variable region gene sequence derived from a human Vκ1-39 orVκ3-20 gene segment, e.g., derived from a rearranged Vκ1-39/Jκ5 orVκ3-20/Jκ1 gene sequence. In one embodiment, wherein the singlerearranged human immunoglobulin light chain variable region genesequence present in the locus is derived from the rearranged Vκ1-39/Jκ5sequence, the substitution of at least one non-histidine codon with ahistidine codon is designed to express a histidine at a positionselected from 105, 106, 108, 111, and a combination thereof. In anotherembodiment, wherein the single rearranged human immunoglobulin lightchain variable region gene sequence present in the locus is derived fromthe rearranged Vκ3-20/Jκ1 sequence, the substitution of at least onenon-histidine codon with a histidine codon is designed to express ahistidine at a position selected from 105, 106, 107, 109, and acombination thereof. In various embodiments, the non-human locidescribed herein may be generated using methods described below formaking a genetically modified non-human animal.

In yet another aspect, provided herein is a method for making anon-human animal that comprises a genetically modified immunoglobulinlight chain locus in its germline, wherein the method comprisesmodifying a genome of a non-human animal to delete or rendernon-functional endogenous immunoglobulin light chain V and J segments inan immunoglobulin light chain locus, and placing in the genome a singlerearranged human light chain variable region gene sequence comprising asubstitution of at least one non-histidine codon with a histidine codon.In one embodiment, such method results in a genetically modifiednon-human animal that comprises a population of B cells enriched forantibodies exhibiting pH-dependent binding to the antigen of interest.In one embodiment, the single rearranged human immunoglobulin lightchain variable region sequence placed in the genome is derived from ahuman Vκ1-39 or Vκ3-20, e.g., a rearranged Vκ1-39/Jκ5 or Vκ3-20/Jκ1 genesequence. Thus, in the embodiment wherein the single rearranged humanimmunoglobulin light chain variable region sequence is derived from arearranged Vκ1-39/Jκ5, the substitution of at least one non-histidinecodon with a histidine codon is designed to express a histidine at aposition selected from 105, 106, 108, 111, and a combination thereof. Inan embodiment wherein the single rearranged human immunoglobulin lightchain variable region sequence is derived from a rearranged Vκ3-20/Jκ1,the substitution of at least one non-histidine codon with a histidinecodon is designed to express a histidine at a position selected from105, 106, 107, 109, and a combination thereof.

In another aspect, provided herein is a method of generating an antibodythat exhibits pH-dependent binding to an antigen of interest comprising(a) generating a mouse described herein (e.g., a mouse that comprises inits germline an immunoglobulin light chain locus that comprises a singlerearranged human immunoglobulin light chain variable region sequencecomprising human V_(L) and J_(L) segment sequences and a substitution ofat least one non-histidine codon with a histidine codon in itsrearranged light chain variable region sequence), (b) immunizing themouse with an antigen of interest, and (c) selecting an antibody thatbinds to the antigen of interest with a desired affinity at a neutral pHwhile displaying reduced binding to the antigen at an acidic pH. In oneembodiment, the method results in a generation of an antibody thatexhibits t_(1/2) at acidic pH and 37° C. of about 2 minutes or less. Inone embodiment, the method results in a generation of an antibody thatdisplays a decrease in dissociative half-life (t_(1/2)) at an acidic pHas compared to neutral pH of at least about 2-fold, at least about3-fold, at least about 4-fold, at least about 5-fold, at least about10-fold, at least about 15-fold, at least about 20-fold, at least about25-fold, or at least about 30-fold.

In other aspects, provided herein are additional methods of generatingan antibody that exhibits pH-dependent binding to an antigen ofinterest. One such method comprises (a) selecting a first antibody thatbinds to an antigen of interest with a desired affinity, (b) modifyingan immunoglobulin light chain nucleotide sequence of the first antibodyto comprise a substitution of at least one non-histidine codon with ahistidine codon, (c) expressing an immunoglobulin heavy chain of thefirst antibody and the modified immunoglobulin light chain in a cell,and (d) selecting a second antibody expressed in the cell that retains adesired affinity for the antigen of interest at neutral pH and displaysreduced binding to the antigen of interest at an acidic pH. In oneembodiment, the immunoglobulin light chain nucleotide sequence of thefirst antibody comprises a single rearranged human immunoglobulin lightchain variable region sequence. In one embodiment, the first antibody isgenerated in a non-human animal, e.g., a mouse, comprising animmunoglobulin light chain sequence derived from a single rearrangedhuman immunoglobulin light chain variable region sequence, and themodification of the immunoglobulin light chain is made in the singlerearranged human immunoglobulin variable region sequence. In oneembodiment, the first antibody is generated in a non-human animal, e.g.,a mouse, further comprising an immunoglobulin heavy chain sequencederived from a repertoire of human V_(H), D_(H), and J_(H) segments. Inone embodiment, the single rearranged human immunoglobulin light chainvariable region sequence is selected from Vκ1-39/Jκ5 and Vκ3-20/Jκ1 genesequence. In an embodiment, wherein the single rearranged humanimmunoglobulin light chain variable region sequence is Vκ1-39/Jκ5, themodification in the immunoglobulin light chain nucleotide sequence ofthe first antibody is made in the CDR3 codon at a position selected from105, 106, 108, 111, and a combination thereof. In an embodiment whereinthe single rearranged human immunoglobulin light chain variable regionsequence is Vκ3-20/Jκ1, the modification in the immunoglobulin lightchain nucleotide sequence of the first antibody is made in the CDR3codon at a position selected from 105, 106, 107, 109, and a combinationthereof.

In one embodiment, the method of generating an antibody that exhibitspH-dependent binding to an antigen of interest described herein resultsin an antibody that displays a decrease in dissociative half-life(t_(1/2)) at an acidic pH as compared to neutral pH of at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold. In oneembodiment, the method of generating the antibody results in an antibodythat exhibits a t_(1/2) at acidic pH and 37° C. of about 2 minutes orless.

Genetically modified immunoglobulin heavy chain loci in the germlinegenome of non-human animals are provided, wherein the immunoglobulinheavy chain loci comprise a genetically modified unrearranged heavychain variable region nucleotide sequence (e.g., one or more geneticallymodified human V_(H), D, and/or J_(H) gene segment), wherein theunrearranged heavy chain variable region nucleotide sequence comprisesan addition of at least one histidine codon or a substitution of atleast one endogenous non-histidine codon with a histidine codon. Invarious embodiments, the genetically modified unrearranged heavy chainvariable region nucleotide sequence comprises at least one histidinecodon in at least one reading frame that encodes an immunoglobulin heavychain variable domain. In various embodiments, the unrearranged heavychain variable region nucleotide sequence comprising the at least onehistidine codon is operably linked to a human or non-human heavy chainconstant region nucleotide sequence (e.g., a heavy chain constant regionnucleotide sequence that encodes an immunoglobulin isotype selected fromIgM, IgD, IgA, IgE, and IgG).

Non-human animals (mammals, e.g., rodents such as mice, rats, orhamsters) are provided that are genetically engineered to containimmunoglobulin heavy chain genomic loci in their germline genome,wherein the genomic loci comprise an unrearranged heavy chain variableregion nucleotide sequence (e.g., one or more genetically modified humanV_(H), D, and/or J_(H) gene segments), wherein the unrearranged heavychain variable region nucleotide sequence comprises an addition of atleast one histidine codon or a substitution of at least one endogenousnon-histidine codon with a histidine codon. In various embodiments, thegenome of the non-human animals comprises a modification (i) thatdeletes or renders nonfunctional all, or substantially all, endogenousimmunoglobulin V_(H), D, and/or J_(H) gene segments (e.g., via insertionof a nucleotide sequence, e.g., an exogenous nucleotide sequence, in theimmunoglobulin locus or via non-functional rearrangement or inversion ofendogenous V_(H), D, and/or J_(H) gene segments); and (ii) thatintroduces an unrearranged human heavy chain variable region nucleotidesequence (e.g., genetically modified human V_(H), D, or J_(H) genesegments), wherein the unrearranged heavy chain variable regionnucleotide sequence comprises an addition of at least one histidinecodon or a substitution of at least one endogenous non-histidine codonwith a histidine codon. In various embodiments, the unrearranged heavychain variable region nucleotide sequence is present at an endogenouslocus (i.e., where the unrearranged heavy chain variable regionnucleotide sequence is located in a wild-type non-human animal) orpresent ectopically (e.g., at a locus different from the endogenousimmunoglobulin heavy chain locus in its genome) or within its endogenouslocus (e.g., within an immunoglobulin variable locus, wherein theendogenous locus is placed or moved to a different location in thegenome). In various embodiments, the immunoglobulin heavy chain variableregion nucleotide sequence is operably linked to a human or non-humanheavy chain constant region nucleotide sequence (e.g., a heavy chainconstant region nucleotide sequence that encodes an immunoglobulinisotype selected from IgM, IgD, IgA, IgE, and IgG).

Genetically modified non-human animals are provided that are capable ofexpressing a genetically modified immunoglobulin heavy variable domaincomprising one or more histidines, wherein the one or more histidinesare not encoded by a germline gene segment of a corresponding wild-typenon-human animal.

Genetically modified non-human animals are provided that comprise a Bcell population that is characterized by rearranged immunoglobulin heavychain variable genes that encode an immunoglobulin heavy chain variabledomain with one or more histidines that are not encoded by a germlinegene segment of a corresponding wild-type non-human animal.

Methods and compositions are provided for making non-human animals thatcomprise a genetically modified immunoglobulin heavy chain variablelocus comprising an unrearranged human heavy chain variable regionnucleotide sequence containing one or more histidine codons in at leastone reading frame that encodes a heavy chain variable domain.

Methods and compositions are provided for non-human animals that makeantigen-binding proteins that exhibit a pH-dependent binding of antigen.Methods and compositions are provided for making non-human animals thathave B cell populations, or antibody populations, that are enriched (ascompared with corresponding wild-type animals) with antigen-bindingproteins that are pH-dependent, e.g., in particular, heavy chainvariable domains, and/or antigen-binding fragments thereof.

In one aspect, a genetically modified immunoglobulin locus in a germlinegenome of a non-human animal is provided comprising an unrearrangedhuman heavy chain variable region nucleotide sequence, wherein theunrearranged heavy chain variable region nucleotide sequence comprisesan addition of least one histidine codon or a substitution of at leastone endogenous non-histidine codon with a histidine codon.

In one embodiment, the non-human animal is a mammal, including a rodent,e.g., a mouse, a rat, or a hamster.

In one embodiment, the added or substituted histidine codon is presentin an immunoglobulin heavy chain gene segment selected from a humanV_(H) gene segment, a human D gene segment, a human J_(H) gene segment,and a combination thereof. In one embodiment, the immunoglobulin heavychain gene segment is selected from a human germline V_(H) gene segment,a human germline D gene segment, a human germline J_(H) gene segment,and a combination thereof.

In one embodiment, the human V gene segment (V_(H)) is selected from thegroup consisting of V_(H)1-2, V_(H)1-3, V_(H)1-8, V_(H)1-18, V_(H)1-24,V_(H)1-45, V_(H)1-46, V_(H)1-58, V_(H)1-69, V_(H)2-5, V_(H)2-26,V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11, V_(H)3-13, V_(H)3-15,V_(H)3-16, V_(H)3-20, V_(H)3-21, V_(H)3-23, V_(H)3-30, V_(H)3-30-3,V_(H)3-30-5, V_(H)3-33, V_(H)3-35, V_(H)3-38, V_(H)3-43, V_(H)3-48,V_(H)3-49, V_(H)3-53, V_(H)3-64, V_(H)3-66, V_(H)3-72, V_(H)3-73,V_(H)3-74, V_(H)4-4, V_(H)4-28, V_(H)4-30-1, V_(H)4-30-2, V_(H)4-30-4,V_(H)4-31, V_(H)4-34, V_(H)4-39, V_(H)4-59, V_(H)4-61, V_(H)5-51,V_(H)6-1, V_(H)7-4-1, V_(H)7-81, and a combination thereof.

In one embodiment, the human D gene segment is selected from the groupconsisting of D1-1, D1-7, D1-14, D1-20, D1-26, D2-2, D2-8, D2-15, D2-21,D3-3, D3-9, D3-10, D3-16, D3-22, D4-4, D4-11, D4-17, D4-23, D5-12, D5-5,D5-18, D5-24, D6-6, D6-13, D6-19, D6-25, D7-27, and a combinationthereof.

In one embodiment, the human J gene segment is selected from the groupconsisting of J_(H)1, J_(H)2, J_(H)3, J_(H)4, J_(H)5, J_(H)6, and acombination thereof.

In one embodiment, the added or substituted histidine codon is presentin the unrearranged heavy chain variable region nucleotide sequence thatencodes an N-terminal region, a loop 4 region, a CDR1, a CDR2, a CDR3,or a combination thereof.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprises 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 ormore, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 ormore, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 ormore, 24 or more, or 25 or more, 26 or more, 27 or more, 28 or more, 29or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more 35or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41or more, 42 or more, 43 or more, 44 or more, 45 or more, 46 or more, 47or more, 48 or more, 49 or more, 50 or more, 51 or more, 52 or more, 53or more, 54 or more, 55 or more, 56 or more, 57 or more, 58 or more, 59or more, 60 or more, or 61 or more of histidine codons.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence is operably linked to a human or non-human heavychain constant region nucleotide sequence that encodes an immunoglobulinisotype selected from IgM, IgD, IgG, IgE, and IgA.

In one embodiment, the human unrearranged immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human ornon-human heavy chain constant region nucleotide sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In oneembodiment, the heavy chain constant region nucleotide sequencecomprises a C_(H)1, a hinge, a C_(H)2, and a C_(H)3(C_(H)1-hinge-C_(H)2-C_(H)3).

In one embodiment, a heavy chain constant region nucleotide sequence ispresent at an endogenous locus (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the heavy chain constant region nucleotide sequencecomprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a modification at position 250 (e.g., E or Q); 250 and 428(e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256(e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433(e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591(e.g., V259I), and a 308F (e.g., V308F) modification; a 433K (e.g.,H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g.,252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g.,T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or308P), wherein the modification increases the affinity of the heavychain constant region amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 252 and 257,wherein the modification increases the affinity of the human C_(H)2amino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 307 and 311,wherein the modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)3 amino acid sequence, wherein the C_(H)3 aminoacid sequence comprises at least one modification between amino acidresidues at positions 433 and 436, wherein the modification increasesthe affinity of the C_(H)3 amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,N434S, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,V259I, V308F, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising an N434A mutation.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M252Y,S254T, T256E, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of T250Q,M248L, or both.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of H433K,N434Y, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises: (1) a first allele, wherein the unrearranged humanimmunoglobulin heavy chain variable region nucleotide sequence asdescribed herein is operably linked to a first heavy chain constantregion nucleotide sequence encoding a first CH₃ amino acid sequence of ahuman IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and(2) a second allele, wherein the unrearranged human immunoglobulin heavychain variable region nucleotide sequence as described herein isoperably linked to a second heavy chain constant region nucleotidesequence encoding a second C_(H)3 amino acid sequence of the human IgGselected from IgG1, IgG2, IgG4, and a combination thereof, and whereinthe second CH₃ amino acid sequence comprises a modification that reducesor eliminates binding for the second CH₃ amino acid sequence to ProteinA (see, for example, US 2010/0331527A1, incorporated by reference hereinin its entirety).

In one embodiment, the second CH₃ amino acid sequence comprises an H95Rmodification (by IMGT exon numbering; H435R by EU numbering). In oneembodiment the second CH₃ amino acid sequence further comprises an Y96Fmodification (by IMGT exon numbering; H436F by EU). In anotherembodiment, the second CH₃ amino acid sequence comprises both an H95Rmodification (by IMGT exon numbering; H435R by EU numbering) and an Y96Fmodification (by IMGT exon numbering; H436F by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG1 and further comprises a mutation selected from the groupconsisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L38M,N384S, K392N, V397M, and V422I by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG2 and further comprises a mutation selected from the groupconsisting of N44S, K52N, and V82I (IMGT: N384S, K392N, and V422I byEU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG4 and further comprises a mutation selected from the groupconsisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT: Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

In one embodiment, the heavy chain constant region amino acid sequenceis a non-human constant region amino acid sequence, and the heavy chainconstant region amino acid sequence comprises one or more of any of thetypes of modifications described above.

In one embodiment, the heavy chain constant region nucleotide sequenceis a human heavy chain constant region amino acid sequence, and thehuman heavy chain constant region amino acid sequence comprises one ormore of any of the types of modifications described above.

In one embodiment, all or substantially all endogenous V_(H), D, andJ_(H) gene segments are deleted from an immunoglobulin heavy chain locusor rendered non-functional (e.g., via insertion of a nucleotide sequence(e.g., an exogenous nucleotide sequence) in the immunoglobulin locus orvia non-functional rearrangement, or inversion, of the endogenous V_(H),D, J_(H) segments). In one embodiment, e.g., about 80% or more, about85% or more, about 90% or more, about 95% or more, about 96% or more,about 97% or more, about 98% or more, or about 99% or more of allendogenous V_(H), D, or J_(H) gene segments are deleted or renderednon-functional. In one embodiment, e.g., at least 95%, 96%, 97%, 98%, or99% of endogenous functional V, D, or J gene segments are deleted orrendered non-functional.

In one embodiment, the genetically modified immunoglobulin heavy chainlocus comprises a modification that deletes or renders, all orsubstantially all, non-functional endogenous V_(H), D, and J_(H) genesegments; and the genetically modified locus comprises an unrearrangedheavy chain variable region nucleotide sequence comprising one or morehuman V_(H), D, and/or J_(H) gene segments having one or more histidinecodons, wherein the unrearranged heavy chain variable region nucleotidesequence is present at an endogenous location (i.e., where thenucleotide sequence is located in a wild-type non-human animal) orpresent ectopically (e.g., at a locus different from the endogenousimmunoglobulin chain locus in its genome, or within its endogenouslocus, e.g., within an immunoglobulin variable locus, wherein theendogenous locus is placed or moved to a different location in thegenome).

In one embodiment, the genetically modified immunoglobulin locuscomprises an endogenous Adam6a gene, Adam6b gene, or both, and thegenetic modification does not affect the expression and/or function ofthe endogenous Adam6a gene, Adam6b gene, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises an ectopically present Adam6a gene, Adam6b gene, or both. Inone embodiment, the Adam6a gene is a non-human Adam6a gene. In oneembodiment, the Adam6a gene is a human Adam6a gene. In one embodiment,the Adam6b gene is a non-human Adam6b gene. In one embodiment, theAdam6b gene is a human Adam6b gene.

In one embodiment, the genetically modified immunoglobulin locus furthercomprises a humanized, unrearranged λ and/or κ light chain variable genesequence. In one embodiment, the humanized, unrearranged λ and/or κlight chain variable gene sequence is operably linked to animmunoglobulin light chain constant region nucleotide sequence selectedfrom a λ light chain constant region nucleotide sequence and a κ lightchain constant region nucleotide sequence. In one embodiment, thehumanized, unrearranged λ light chain variable region nucleotidesequence is operably linked to a λ light chain constant regionnucleotide sequence. In one embodiment, the λ light chain constantregion nucleotide sequence is a mouse, rat, or human sequence. In oneembodiment, the humanized, unrearranged κ light chain variable regionnucleotide sequence is operably linked to a κ light chain constantregion nucleotide sequence. In one embodiment, the κ light chainconstant region nucleotide sequence is a mouse, rat, or human sequence.

In one embodiment, the genetically modified immunoglobulin locuscomprises an unrearranged light chain variable gene sequence thatcontains at least one modification that introduces at least onehistidine codon in at least one reading frame encoding a light chainvariable domain. In one embodiment, the genetically modifiedimmunoglobulin locus comprises a rearranged (e.g., rearranged λ or κ V/Jsequence) sequence that comprises one, two, three, or four codons forhistidine in a light chain CDR. In one embodiment, the CDR is a selectedfrom a CDR1, CDR2, CDR3, and a combination thereof. In one embodiment,the unrearranged or rearranged light chain variable region nucleotidesequence is an unrearranged or rearranged human λ or κ light chainvariable region nucleotide sequence. In one embodiment, the unrearrangedor rearranged human λ or κ light chain variable region nucleotidesequence is present at an endogenous mouse immunoglobulin light chainlocus. In one embodiment, the mouse immunoglobulin light chain locus isa mouse κ locus. In one embodiment, the mouse immunoglobulin light chainlocus is a mouse λ locus.

In one embodiment, the genetically modified immunoglobulin locus asdescribed herein is present in an immunoglobulin heavy chain locus of amouse. In one embodiment, the genetically modified immunoglobulin locusis present in a humanized immunoglobulin heavy chain locus in aVELOCIMMUNE® mouse.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein exhibits a weaker antigen bindingat an acidic environment (e.g., at a pH of about 5.5 to about 6.0) thana corresponding wild-type heavy chain variable domain without thegenetic modification.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises a B cell population that, upon stimulationwith an antigen of interest, is capable of producing antigen-bindingproteins, e.g., antibodies, comprising a heavy chain variable domainwith one or more histidine residues. The antigen-binding proteins asdescribed herein when administered into a subject, exhibits an increasedserum half-life over a corresponding wild-type antigen-binding protein,which possesses a similar or sufficiently similar amino acid sequencethat encodes the heavy chain variable domain but does not comprise ahistidine residue in the heavy chain variable domain. In someembodiments, the antigen-binding protein described herein exhibits anincreased serum half-life that is at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold higher than the corresponding wild-type antigen-binding protein,which possesses a similar or sufficiently similar amino acid sequencethat encodes the heavy chain variable domain but does not comprise ahistidine residue in the heavy chain variable domain.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modificationdescribed herein.

In one aspect, a genetically modified immunoglobulin locus in a germlinegenome of a non-human animal is provided comprising an unrearrangedhuman heavy chain variable region nucleotide sequence, wherein the humanunrearranged heavy chain variable region nucleotide sequence comprises asubstitution of at least one endogenous non-histidine codon with ahistidine codon.

In one embodiment, the non-human animal is a mammal, including a rodent,e.g., a mouse, a rat, or a hamster.

In one embodiment, 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 ormore, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 ormore, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 ormore, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 ormore, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 ormore, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 ormore, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, 48 ormore, 49 or more, 50 or more, 51 or more, 52 or more, 53 or more, 54 ormore, 55 or more, 56 or more, 57 or more, 58 or more, 59 or more, 60 ormore, or 61 or more of the endogenous non-histidine codons are replacedwith histidine codons.

In one embodiment, the endogenous non-histone codon encodes the aminoacid selected from Y, N, D, Q, S, W, and R.

In one embodiment, the endogenous non-histidine codon that issubstituted by the histidine codon is present in an unrearranged heavychain variable region nucleotide sequence that encodes an immunoglobulinvariable domain selected from an N-terminal region, a loop 4 region, aCDR1, a CDR2, a CDR3, a combination thereof.

In one embodiment, the substituted histidine codon is present in anunrearranged heavy chain variable region nucleotide sequence thatencodes a complementary determining region (CDR) selected from a CDR1, aCDR2, a CDR3, and a combination thereof.

In one embodiment, the substituted histidine codon is present in anunrearranged heavy chain variable region nucleotide sequence thatencodes a frame region (FR) selected from FR1, FR2, FR3, FR4, and acombination thereof.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprises a genetically modified human V_(H) genesegment, wherein one or more endogenous non-histidine codon in at leastone reading frame of the human V_(H) gene segment has been replaced witha histidine codon.

In one embodiment, the human unrearranged heavy chain variable regionnucleotide sequence comprises a modification that replaces at least oneendogenous non-histidine codon of a human V_(H) gene segment with ahistidine codon, wherein the human V_(H) gene segment is selected fromthe group consisting of V_(H)1-2, V_(H)1-3, V_(H)1-8, V_(H)1-18,V_(H)1-24, V_(H)1-45, V_(H)1-46, V_(H)1-58, V_(H)1-69, V_(H)2-5,V_(H)2-26, V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11, V_(H)3-13,V_(H)3-15, V_(H)3-16, V_(H)3-20, V_(H)3-21, V_(H)3-23, V_(H)3-30,V_(H)3-30-3, V_(H)3-30-5, V_(H)3-33, V_(H)3-35, V_(H)3-38, V_(H)3-43,V_(H)3-48, V_(H)3-49, V_(H)3-53, V_(H)3-64, V_(H)3-66, V_(H)3-72,V_(H)3-73, V_(H)3-74, V_(H)4-4, V_(H)4-28, V_(H)4-30-1, V_(H)4-30-2,V_(H)4-30-4, V_(H)4-31, V_(H)4-34, V_(H)4-39, V_(H)4-59, V_(H)4-61,V_(H)5-51, V_(H)6-1, V_(H)7-4-1, V_(H)7-81, and a combination thereof.

In one embodiment, the human unrearranged heavy chain variable regionnucleotide sequence comprises a genetically modified human J_(H) genesegment, wherein one or more endogenous non-histidine codon in at leastone reading frame of the human J_(H) gene segment has been replaced witha histidine codon.

In one embodiment, the human unrearranged heavy chain variable regionnucleotide sequence comprises a modification that replaces at least oneendogenous non-histidine codon of a human J_(H) segment with a histidinecodon, wherein the human J_(H) gene segment is selected from the groupconsisting of J_(H)1, J_(H)2, J_(H)3, J_(H)4, J_(H)5, J_(H)6, and acombination thereof.

In one embodiment, the substituted histidine codon is present in a heavychain variable region nucleotide sequence that encodes part of a CDR3.In one embodiment, the part of CDR3 comprises an amino acid sequencederived from a reading frame of a genetically modified human D genesegment comprising a modification that replaces at least one endogenousnon-histidine codon in the reading frame with a histidine codon.

In one embodiment, the endogenous non-histidine codon that issubstituted with a histidine codon encodes the amino acid selected fromY, N, D, Q, S, W, and R.

In one embodiment, the substituted histidine codon is present in atleast one reading frame of the human D gene segment that is mostfrequently observed in VELOCIMMUNE® humanized immunoglobulin mice.

In one embodiment, the reading frame of the genetically modified human Dgene segment that encodes part of CDR3 is selected from a hydrophobicframe, a stop frame, and a hydrophilic frame.

In one embodiment, the reading frame is a hydrophobic frame of a human Dgene segment.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D1-1 (GTTGT; SEQ ID NO: 88), D1-7(GITGT; SEQ ID NO: 89), D1-20 (GITGT; SEQ ID NO: 89), and D1-26 (GIVGAT;SEQ ID NO: 90), and the human D gene segment further comprises amodification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D2-2 (DIVVVPAAI; SEQ ID NO: 92),D2-8 (DIVLMVYAI; SEQ ID NO: 94), D2-15 (DIVVVVAAT; SEQ ID NO: 95), andD2-21 (HIVVVTAI; SEQ ID NO: 97), and the human D gene segment furthercomprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D3-3 (ITIFGVVII; SEQ ID NO: 98),D3-9 (ITIF*LVII; SEQ ID NO: 99, SEQ ID NO:100), D3-10 (ITMVRGVII; SEQ IDNO:101), D3-16 (IMITFGGVIVI; SEQ ID NO:102), and D3-22 (ITMIWVIT; SEQ IDNO:103), and the human D gene segment further comprises a modificationthat replaces at least one endogenous codon in the nucleotide sequencewith a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D4-4 (TTVT; SEQ ID NO: 105), D4-11(TTVT; SEQ ID NO:105), D4-17 (TTVT; SEQ ID NO:105), D4-23 (TTVVT; SEQ IDNO: 106) and the human D gene segment further comprises a modificationthat replaces at least one endogenous non-histidine codon in thenucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D5-5 (VDTAMV; SEQ ID NO: 107),D5-12 (VDIVATI; SEQ ID NO: 108), D5-18 (VDTAMV; SEQ ID NO:107), andD5-24 (VEMATI; SEQ ID NO:109), and the human D gene segment furthercomprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D6-6 (SIAAR; SEQ ID NO: 111),D6-13 (GIAAAG; SEQ ID NO: 113), and D6-19 (GIAVAG; SEQ ID NO:115), andthe human D gene segment further comprises a modification that replacesat least one endogenous non-histidine codon in the nucleotide sequencewith a histidine codon.

In one embodiment, the hydrophobic frame comprises a nucleotide sequencethat encodes human D7-27 (LTG), and the human D gene segment furthercomprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the reading frame is a stop reading frame of a humanD gene segment.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D1-1 (VQLER; SEQ ID NO:8),D1-7(V*LEL), D1-20(V*LER), D1-26 (V*WELL; SEQ ID NO: 12), and the humanD gene segment further comprises a modification that replaces at leastone endogenous non-histidine codon in the nucleotide sequence with ahistidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D2-2 (RIL**YQLLY; SEQ ID NO:14),D2-8 (RILY*WCMLY; SEQ ID NO:16 and SEQ ID NO: 17), D2-15 (RIL*WW*LLL),and D2-21 (SILWW*LLF; SEQ ID NO:19), and the human D gene segmentfurther comprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D3-3 (VLRFLEWLLY; SEQ ID NO:21),D3-9 (VLRYFDWLL*; SEQ ID NO:23), D3-10 (VLLWFGELL*; SEQ ID NO:25), D3-16(VL*LRLGELSLY; SEQ ID NO:27), and D3-22 (VLL***WLLL; SEQ ID NO:29), andthe human D gene segment comprises a modification that replaces at leastone endogenous non-histidine codon in the nucleotide sequence with ahistidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D4-4 (*LQ*L), D4-11 (*LQ*L), D4-17(*LR*L), and D4-23 (*LRW*L), and the human D gene segment comprises amodification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D5-5 (WIQLWL; SEQ ID NO:35); D5-12(WI*WLRL; SEQ ID NO:37), D5-18 (WIQLWL; SEQ ID NO:35), and D5-24(*RWLQL; SEQ ID NO:39), and the human D gene segment comprises amodification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D6-6 (V*QLV), D6-13 (V*QQLV; SEQID NO:41), and D6-19 (V*QWLV; SEQ ID NO:43), and the human D genesegment further comprises a modification that replaces at least oneendogenous non-histidine codon in the nucleotide sequence with ahistidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes D7-27 (*LG), and the humanD gene segment further comprises a modification that replaces at leastone endogenous codon of the human D gene segment in the nucleotidesequence with a histidine codon.

In one embodiment, the reading frame is a hydrophilic frame of a human Dgene segment.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D1-1 (YNWND; SEQ ID NO: 45), D1-7(YNWNY; SEQ ID NO: 47), D1-20 (YNWND; SEQ ID NO: 45), and D1-26 (YSGSYY;SEQ ID NO:49), and the human D gene segment further comprises amodification that replaces at least one endogenous codon in thenucleotide sequence with a histidine codon. In one embodiment, thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 46, SEQID NO: 48, SEQ ID NO: 50, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D2-2 (GYCSSTSCYT; SEQ ID NO:51),D2-8 (GYCTNGVCYT; SEQ ID NO: 53), D2-15 (GYCSGGSCYS; SEQ ID NO:55), andD2-21 (AYCGGDCYS; SEQ ID NO:57), and the human D gene segment furthercomprises a modification that replaces at least one endogenous codon inthe nucleotide sequence with a histidine codon. In one embodiment, thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 52, SEQID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D3-3 (YYDFWSGYYT; SEQ ID NO:59),D3-9 (YYDILTGYYN; SEQ ID NO:61), D3-10 (YYYGSGSYYN; SEQ ID NO:63), D3-16(YYDYVWGSYRYT; SEQ ID NO:65), and D3-22 (YYYDSSGYYY; SEQ ID NO:67), andthe human D gene segment further comprises a modification that replacesat least one endogenous codon in the nucleotide sequence with ahistidine codon. In one embodiment, the hydrophilic frame comprises anucleotide sequence encodes the amino acid sequence selected from thegroup consisting of SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ IDNO: 66, SEQ ID NO: 68, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D4-4 (DYSNY; SEQ ID NO:69), D4-11(DYSNY; SEQ ID NO:69), D4-17 (DYGDY; SEQ ID NO:71), and D4-23 (DYGGNS;SEQ ID NO:73), and the human D gene segment comprises a modificationthat replaces at least one endogenous codon in the nucleotide sequencewith a histidine codon. In one embodiment, the hydrophilic framecomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 72, SEQID NO: 74, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D5-5 (GYSYGY; SEQ ID NO:75), D5-12(GYSGYDY; SEQ ID NO:77), D5-18 (GYSYGY; SEQ ID NO:75), and D5-24(RDGYNY; SEQ ID NO:79), and the human D gene segment further comprises amodification that replaces at least one endogenous codon in thenucleotide sequence with a histidine codon. In one embodiment, thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 76, SEQID NO: 78, SEQ ID NO: 80, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D6-6 (EYSSSS; SEQ ID NO: 81),D6-13 (GYSSSWY; SEQ ID NO:83), and D6-19 (GYSSGWY; SEQ ID NO:85), andthe human D gene segment further comprises a modification that replacesat least one endogenous codon in the nucleotide sequence with ahistidine codon. In one embodiment, the hydrophilic frame comprises anucleotide sequence that encodes the amino acid sequence selected fromthe group consisting of SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQID NO: 76, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes D7-27 (NWG), and the humanD gene segment further comprises a modification that replaces at leastone endogenous codon in the nucleotide sequence a histidine codon.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO:68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ IDNO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, anda combination thereof.

In one embodiment, the human unrearranged immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human ornon-human heavy chain constant region nucleotide sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In oneembodiment, the heavy chain constant region nucleotide sequencecomprises a C_(H)1, a hinge, a C_(H)2, and a C_(H)3(C_(H)1-hinge-C_(H)2-C_(H)3).

In one embodiment, a heavy chain constant region nucleotide sequence ispresent at an endogenous locus (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the heavy chain constant region nucleotide sequencecomprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a modification at position 250 (e.g., E or Q); 250 and 428(e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256(e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433(e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591(e.g., V259I), and a 308F (e.g., V308F) modification; a 433K (e.g.,H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g.,252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g.,T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or308P), wherein the modification increases the affinity of the heavychain constant region amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 252 and 257,wherein the modification increases the affinity of the human C_(H)2amino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 307 and 311,wherein the modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)3 amino acid sequence, wherein the C_(H)3 aminoacid sequence comprises at least one modification between amino acidresidues at positions 433 and 436, wherein the modification increasesthe affinity of the C_(H)3 amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,N434S, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,V259I, V308F, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising an N434A mutation.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M252Y,S254T, T256E, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of T250Q,M248L, or both.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of H433K,N434Y, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises: (1) a first allele, wherein the unrearranged humanimmunoglobulin heavy chain variable region nucleotide sequence asdescribed herein is operably linked to a first heavy chain constantregion nucleotide sequence encoding a first CH₃ amino acid sequence of ahuman IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and(2) a second allele, wherein the unrearranged human immunoglobulin heavychain variable region nucleotide sequence as described herein isoperably linked to a second heavy chain constant region nucleotidesequence encoding a second C_(H)3 amino acid sequence of the human IgGselected from IgG1, IgG2, IgG4, and a combination thereof, and whereinthe second CH₃ amino acid sequence comprises a modification that reducesor eliminates binding for the second CH₃ amino acid sequence to ProteinA (see, for example, US 2010/0331527A1, incorporated by reference hereinin its entirety).

In one embodiment, the second CH₃ amino acid sequence comprises an H95Rmodification (by IMGT exon numbering; H435R by EU numbering). In oneembodiment the second CH₃ amino acid sequence further comprises an Y96Fmodification (by IMGT exon numbering; H436F by EU). In anotherembodiment, the second CH₃ amino acid sequence comprises both an H95Rmodification (by IMGT exon numbering; H435R by EU numbering) and an Y96Fmodification (by IMGT exon numbering; H436F by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG1 and further comprises a mutation selected from the groupconsisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L38M,N384S, K392N, V397M, and V422I by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG2 and further comprises a mutation selected from the groupconsisting of N44S, K52N, and V82I (IMGT: N384S, K392N, and V422I byEU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG4 and further comprises a mutation selected from the groupconsisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT: Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

In one embodiment, the heavy chain constant region amino acid sequenceis a non-human constant region amino acid sequence, and the heavy chainconstant region amino acid sequence comprises one or more of any of thetypes of modifications described above.

In one embodiment, the heavy chain constant region nucleotide sequenceis a human heavy chain constant region amino acid sequence, and thehuman heavy chain constant region amino acid sequence comprises one ormore of any of the types of modifications described above.

In one embodiment, all or substantially all endogenous V_(H), D, andJ_(H) gene segments are deleted from an immunoglobulin heavy chain locusor rendered non-functional (e.g., via insertion of a nucleotide sequence(e.g., an exogenous nucleotide sequence) in the immunoglobulin locus orvia non-functional rearrangement, or inversion, of the endogenous V_(H),D, J_(H) segments). In one embodiment, e.g., about 80% or more, about85% or more, about 90% or more, about 95% or more, about 96% or more,about 97% or more, about 98% or more, or about 99% or more of allendogenous V_(H), D, or J_(H) gene segments are deleted or renderednon-functional. In one embodiment, e.g., at least 95%, 96%, 97%, 98%, or99% of endogenous functional V, D, or J gene segments are deleted orrendered non-functional.

In one embodiment, the genetically modified locus comprises amodification that deletes or renders non-functional all or substantiallyall endogenous V_(H), D, and J_(H) gene segments; and the genomic locuscomprises the genetically modified, unrearranged human heavy chainvariable region nucleotide sequence comprising a substitution of atleast one endogenous non-histidine codon with a histidine codon in atleast one reading frame. In one embodiment, the genetically modified,unrearranged immunoglobulin heavy chain variable gene sequence ispresent at an endogenous location (i.e., where the nucleotide sequenceis located in a wild-type non-human animal) or present ectopically(e.g., at a locus different from the endogenous immunoglobulin chainlocus in its genome or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the genetically modified locus comprises anendogenous Adam6a gene, Adam6b gene, or both, and the geneticmodification does not affect the expression and/or function of theendogenous Adam6a gene, Adam6b gene, or both.

In one embodiment, the genetically modified locus comprises anectopically present Adam6a gene, Adam6b gene, or both. In oneembodiment, the Adam6a gene is a non-human Adam6a gene. In oneembodiment, the Adam6a gene is a mouse Adam6a gene. In one embodiment,the Adam6a gene is a human Adam6a gene. In one embodiment, the Adam6bgene is a non-human Adam6b gene. In one embodiment, the Adam6b gene is amouse Adam6b gene. In one embodiment, the Adam6b gene is a human Adam6bgene.

In one embodiment, the genetically modified immunoglobulin locus furthercomprises a humanized, unrearranged λ and/or κ light chain variable genesequence. In one embodiment, the humanized, unrearranged λ and/or κlight chain variable gene sequence is operably linked to animmunoglobulin light chain constant region nucleotide sequence selectedfrom a λ light chain constant region nucleotide sequence and a κ lightchain constant region nucleotide sequence. In one embodiment, thehumanized, unrearranged λ light chain variable region nucleotidesequence is operably linked to a λ light chain constant regionnucleotide sequence. In one embodiment, the λ light chain constantregion nucleotide sequence is a mouse, rat, or human sequence. In oneembodiment, the humanized, unrearranged κ light chain variable regionnucleotide sequence is operably linked to a κ light chain constantregion nucleotide sequence. In one embodiment, the κ light chainconstant region nucleotide sequence is a mouse, rat, or human sequence.

In one embodiment, the genetically modified immunoglobulin locuscomprises an unrearranged light chain variable gene sequence thatcontains at least one modification that introduces at least onehistidine codon in at least one reading frame encoding a light chainvariable domain. In one embodiment, the genetically modifiedimmunoglobulin locus comprises a rearranged (e.g., a rearranged λ or κV/J sequence) sequence that comprises one, two, three, or four codonsfor histidine in a light chain CDR. In one embodiment, the CDR is aselected from a CDR1, CDR2, CDR3, and a combination thereof. In oneembodiment, the unrearranged or rearranged light chain variable regionnucleotide sequence is an unrearranged or rearranged human λ or κ lightchain variable region nucleotide sequence. In one embodiment, theunrearranged or rearranged human λ or κ light chain variable regionnucleotide sequence is present at an endogenous mouse immunoglobulinlight chain locus. In one embodiment, the mouse immunoglobulin lightchain locus is a mouse κ locus. In one embodiment the mouseimmunoglobulin light chain locus is a mouse λ locus.

In one embodiment, the genetically modified immunoglobulin locus asdescribed herein is present in an immunoglobulin heavy chain locus of amouse. In one embodiment, the genetically modified immunoglobulin locusis present in a humanized immunoglobulin heavy chain locus in aVELOCIMMUNE® mouse.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein exhibits a weaker antigen bindingat an acidic environment (e.g., at a pH of about 5.5 to about 6.0) thana corresponding wild-type heavy chain variable domain without thegenetic modification described herein.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification.

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises a B cell population that, upon stimulationwith an antigen of interest, is capable of producing antigen-bindingproteins, e.g., antibodies, comprising a heavy chain variable domaincomprising one or more histidine residues. The antigen-binding proteinsas described herein when administered into a subject, exhibits anincreased serum half-life over a corresponding wild-type antigen-bindingprotein, which possesses a similar or sufficiently similar amino acidsequence that encodes the heavy chain variable domain but does notcomprise a histidine residue in the heavy chain variable domain. In someembodiments, the antigen-binding protein described herein exhibits anincreased serum half-life that is at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold higher than the corresponding wild-type antigen-binding protein,which possesses a similar or sufficiently similar amino acid sequencethat encodes the heavy chain variable domain but does not comprise ahistidine residue in the heavy chain variable domain.

In one aspect, a genetically modified immunoglobulin locus of anon-human animal comprising a human V_(H), D, and J_(H) gene segment isprovided, wherein at least one of the human D gene segment has beeninverted 5′ to 3′ with respect to a corresponding wild-type sequence,and wherein at least one reading frame of the inverted human D genesegment comprises a histidine codon.

In one embodiment, the non-human animal is a mammal, including a rodent,e.g., a mouse, a rat, or a hamster

In one embodiment, the genetically modified immunoglobulin locus ispresent in a germline genome.

In one embodiment, the genetically modified immunoglobulin locus encodesan immunoglobulin heavy chain variable domain comprising one or more, 2or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 ormore, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 ormore, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 ormore, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 ormore, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 ormore, 33 or more, or 34 or more of histidine residues.

In one embodiment, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least nine, atleast ten, at least eleven, at least twelve, at least thirteen, at leastfourteen, at least fifteen, at least sixteen, at least seventeen, atleast eighteen, at least nineteen, at least twenty, at least twenty one,at least twenty two, at least twenty three, at least twenty four, or allor substantially all of functional human D gene segments have invertedorientation with respect to corresponding wild type sequences.

In one embodiment, all or substantially all of endogenous immunoglobulinV_(H), D, J_(H) gene segments are deleted from the immunoglobulin heavychain locus or rendered non-functional (e.g., via insertion of anucleotide sequence, e.g., exogenous nucleotide sequence, in theimmunoglobulin locus or via non-functional rearrangement or inversion ofall, or substantially all, endogenous immunoglobulin V_(H), D, J_(H)segments), and the genetically modified immunoglobulin locus comprises ahuman V_(H), D, and J_(H) gene segments, wherein at least one of thehuman D gene segment is present in an inverted orientation with respectto a corresponding wild type sequence, and wherein at least one readingframe in the inverted human D gene segment comprises at least onehistidine codon.

In one embodiment, the inverted human D gene segment is operably linkedto a human V_(H) gene segment, and/or human J_(H) gene segment

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence isselected from the group consisting of D1-1, D1-7, D1-20, D1-26, D2-2,D2-8, D2-15, D2-21, D3-3, D3-9, D3-10, D3-16, D3-22, D4-4, D4-11, D4-17,D4-23, D5-5, D5-12, D5-18, D5-24, D6-6, D6-13, D6-19, D7-27, and acombination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD1 gene segment selected from the group consisting of D1-1, D1-7, D1-20,D1-26, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative a corresponding wild type sequence is a D2gene segment selected from the group consisting of D2-2, D2-8, D2-15,D2-21, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD3 gene segment selected from the group consisting of D3-3, D3-9, D3-10,D3-16, D3-22, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD4 gene segment selected from the group consisting of D4-4, D4-11,D4-17, D4-23, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD5 gene segment selected from the group consisting of D5-5, D5-12,D5-18, D5-24, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD6 gene segment selected from the group consisting of D6-6, D6-13,D6-19, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence isD7-27.

In one embodiment, the reading frame of the human D gene segment isselected from a stop reading frame, a hydrophilic reading frame, and ahydrophobic reading frame, and at least one reading frame of theinverted human D gene segment comprises a histidine codon.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprising the inverted human D gene segment isoperably linked to a human or non-human heavy chain constant regionnucleotide sequence that encodes an immunoglobulin isotype selected fromIgM, IgD, IgG, IgE, and IgA.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprising the inverted human D gene segment isoperably linked to a human or non-human heavy chain constant regionnucleotide sequence that encodes an immunoglobulin isotype selected fromIgM, IgD, IgG, IgE, and IgA.

In one embodiment, the human unrearranged immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human ornon-human heavy chain constant region nucleotide sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In oneembodiment, the heavy chain constant region nucleotide sequencecomprises a C_(H)1, a hinge, a C_(H)2, and a C_(H)3(C_(H)1-hinge-C_(H)2-C_(H)3).

In one embodiment, a heavy chain constant region nucleotide sequence ispresent at an endogenous locus (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the heavy chain constant region nucleotide sequencecomprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a modification at position 250 (e.g., E or Q); 250 and 428(e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256(e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433(e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591(e.g., V259I), and a 308F (e.g., V308F) modification; a 433K (e.g.,H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g.,252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g.,T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or308P), wherein the modification increases the affinity of the heavychain constant region amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 252 and 257,wherein the modification increases the affinity of the human C_(H)2amino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 307 and 311,wherein the modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)3 amino acid sequence, wherein the C_(H)3 aminoacid sequence comprises at least one modification between amino acidresidues at positions 433 and 436, wherein the modification increasesthe affinity of the C_(H)3 amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,N434S, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,V259I, V308F, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising an N434A mutation.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M252Y,S254T, T256E, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of T250Q,M248L, or both.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of H433K,N434Y, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises: (1) a first allele, wherein the unrearranged humanimmunoglobulin heavy chain variable region nucleotide sequence asdescribed herein is operably linked to a first heavy chain constantregion nucleotide sequence encoding a first CH₃ amino acid sequence of ahuman IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and(2) a second allele, wherein the unrearranged human immunoglobulin heavychain variable region nucleotide sequence as described herein isoperably linked to a second heavy chain constant region nucleotidesequence encoding a second C_(H)3 amino acid sequence of the human IgGselected from IgG1, IgG2, IgG4, and a combination thereof, and whereinthe second CH₃ amino acid sequence comprises a modification that reducesor eliminates binding for the second CH₃ amino acid sequence to ProteinA (see, for example, US 2010/0331527A1, incorporated by reference hereinin its entirety).

In one embodiment, the second CH₃ amino acid sequence comprises an H95Rmodification (by IMGT exon numbering; H435R by EU numbering). In oneembodiment the second CH₃ amino acid sequence further comprises an Y96Fmodification (by IMGT exon numbering; H436F by EU). In anotherembodiment, the second CH₃ amino acid sequence comprises both an H95Rmodification (by IMGT exon numbering; H435R by EU numbering) and an Y96Fmodification (by IMGT exon numbering; H436F by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG1 and further comprises a mutation selected from the groupconsisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L38M,N384S, K392N, V397M, and V422I by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG2 and further comprises a mutation selected from the groupconsisting of N44S, K52N, and V82I (IMGT: N384S, K392N, and V422I byEU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG4 and further comprises a mutation selected from the groupconsisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT: Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

In one embodiment, the heavy chain constant region amino acid sequenceis a non-human constant region amino acid sequence, and the heavy chainconstant region amino acid sequence comprises one or more of any of thetypes of modifications described above.

In one embodiment, the heavy chain constant region nucleotide sequenceis a human heavy chain constant region amino acid sequence, and thehuman heavy chain constant region amino acid sequence comprises one ormore of any of the types of modifications described above.

In one embodiment, all or substantially all endogenous V_(H), D, andJ_(H) gene segments are deleted from an immunoglobulin heavy chain locusor rendered non-functional (e.g., via insertion of a nucleotide sequence(e.g., an exogenous nucleotide sequence) in the immunoglobulin locus orvia non-functional rearrangement, or inversion, of the endogenous V_(H),D, J_(H) segments). In one embodiment, e.g., about 80% or more, about85% or more, about 90% or more, about 95% or more, about 96% or more,about 97% or more, about 98% or more, or about 99% or more of allendogenous V_(H), D, or J_(H) gene segments are deleted or renderednon-functional. In one embodiment, e.g., at least 95%, 96%, 97%, 98%, or99% of endogenous functional V, D, or J gene segments are deleted orrendered non-functional.

In one embodiment, the genetically modified immunoglobulin heavy chainlocus comprises a modification that deletes or renders, all orsubstantially all, non-functional endogenous V_(H), D, and J_(H) genesegments; and the genetically modified locus comprises an unrearrangedheavy chain variable region nucleotide sequence comprising at least oneinverted human D gene segment as described herein wherein theunrearranged heavy chain variable region nucleotide sequence is presentat an endogenous location (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the genetically modified immunoglobulin locuscomprises an endogenous Adam6a gene, Adam6b gene, or both, and thegenetic modification does not affect the expression and/or function ofthe endogenous Adam6a gene, Adam6b gene, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises an ectopically present Adam6a gene, Adam6b gene, or both. Inone embodiment, the Adam6a gene is a non-human Adam6a gene. In oneembodiment, the Adam6a gene is a mouse Adam6a gene. In one embodiment,the Adam6a gene is a human Adam6a gene. In one embodiment, the Adam6bgene is a non-human Adam6b gene. In one embodiment, the Adam6b gene is amouse Adam6b gene. In one embodiment, the Adam6b gene is a human Adam6bgene.

In one embodiment, the genetically modified immunoglobulin locus furthercomprises a humanized, unrearranged λ and/or κ light chain variable genesequence. In one embodiment, the humanized, unrearranged λ and/or κlight chain variable gene sequence is operably linked to animmunoglobulin light chain constant region nucleotide sequence selectedfrom a λ light chain constant region nucleotide sequence and a κ lightchain constant region nucleotide sequence. In one embodiment, thehumanized, unrearranged λ light chain variable region nucleotidesequence is operably linked to a λ light chain constant regionnucleotide sequence. In one embodiment, the λ light chain constantregion nucleotide sequence is a mouse, rat, or human sequence. In oneembodiment, the humanized, unrearranged κ light chain variable regionnucleotide sequence is operably linked to a κ light chain constantregion nucleotide sequence. In one embodiment, the κ light chainconstant region nucleotide sequence is a mouse, rat, or human sequence.

In one embodiment, the genetically modified immunoglobulin locuscomprises an unrearranged light chain variable gene sequence thatcontains at least one modification that introduces at least onehistidine codon in at least one reading frame encoding a light chainvariable domain. In one embodiment, the genetically modifiedimmunoglobulin locus comprises a rearranged (e.g., a rearranged λ or κV/J sequence) sequence that comprises one, two, three, or four codonsfor histidine in a light chain CDR. In one embodiment, the CDR is aselected from a CDR1, CDR2, CDR3, and a combination thereof. In oneembodiment, the unrearranged or rearranged light chain variable regionnucleotide sequence is an unrearranged or rearranged human λ or κ lightchain variable region nucleotide sequence. In one embodiment, theunrearranged or rearranged human λ or κ light chain variable regionnucleotide sequence is present at an endogenous mouse immunoglobulinlight chain locus. In one embodiment, the mouse immunoglobulin lightchain locus is a mouse κ locus. In one embodiment, the mouseimmunoglobulin light chain locus is a mouse immunoglobulin light chainlocus is a mouse λ locus.

In one embodiment, the genetically modified immunoglobulin locus asdescribed herein is present in an immunoglobulin heavy chain locus of amouse. In one embodiment, the genetically modified immunoglobulin locusis present in a humanized immunoglobulin heavy chain locus in aVELOCIMMUNE® mouse.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein exhibits a weaker antigen bindingat an acidic environment (e.g., at a pH of about 5.5 to about 6.0) thana corresponding wild-type heavy chain variable domain without thegenetic modification.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification.

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises a B cell population that, upon stimulationwith an antigen of interest, is capable of producing antigen-bindingproteins, e.g., antibodies, comprising a heavy chain variable domaincomprising one or more histidine residues. The antigen-binding proteinsas described herein when administered into a subject, exhibits anincreased serum half-life over a corresponding wild-type antigen-bindingprotein, which possesses a similar or sufficiently similar amino acidsequence that encodes the heavy chain variable domain but does notcomprise a histidine residue in the heavy chain variable domain. In someembodiments, the antigen-binding protein described herein exhibits anincreased serum half-life that is at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold higher than the corresponding wild-type antigen-binding protein,which possesses a similar or sufficiently similar amino acid sequencethat encodes the heavy chain variable domain but does not comprise ahistidine residue in the heavy chain variable domain.

In one aspect, a non-human animal is provided comprising in its germlinegenome a genetically modified immunoglobulin locus comprising anunrearranged human heavy chain variable region nucleotide sequence,wherein the unrearranged heavy chain variable region nucleotide sequencecomprises an addition of least one histidine codon or a substitution ofat least one endogenous non-histidine codon with a histidine codon.

In one embodiment, the non-human animal is a mammal, including a rodent,e.g., a mouse, a rat, or a hamster.

In one embodiment, the added or substituted histidine codon is presentin an immunoglobulin heavy chain gene segment selected from a humanV_(H) gene segment, a human D gene segment, a human J_(H) gene segment,and a combination thereof. In one embodiment, the immunoglobulin heavychain gene segment is selected from a human germline V_(H) gene segment,a human germline D gene segment, a human germline J_(H) gene segment,and a combination thereof.

In one embodiment, the human V_(H) gene segment is selected from thegroup consisting of V_(H)1-2, V_(H)1-3, V_(H)1-8, V_(H)1-18, V_(H)1-24,V_(H)1-45, V_(H)1-46, V_(H)1-58, V_(H)1-69, V_(H)2-5, V_(H)2-26,V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11, V_(H)3-13, V_(H)3-15,V_(H)3-16, V_(H)3-20, V_(H)3-21, V_(H)3-23, V_(H)3-30, V_(H)3-30-3,V_(H)3-30-5, V_(H)3-33, V_(H)3-35, V_(H)3-38, V_(H)3-43, V_(H)3-48,V_(H)3-49, V_(H)3-53, V_(H)3-64, V_(H)3-66, V_(H)3-72, V_(H)3-73,V_(H)3-74, V_(H)4-4, V_(H)4-28, V_(H)4-30-1, V_(H)4-30-2, V_(H)4-30-4,V_(H)4-31, V_(H)4-34, V_(H)4-39, V_(H)4-59, V_(H)4-61, V_(H)5-51,V_(H)6-1, V_(H)7-4-1, V_(H)7-81, and a combination thereof.

In one embodiment, the human D gene segment is selected from the groupconsisting of D1-1, D1-7, D1-14, D1-20, D1-26, D2-2, D2-8, D2-15, D2-21,D3-3, D3-9, D3-10, D3-16, D3-22, D4-4, D4-11, D4-17, D4-23, D5-12, D5-5,D5-18, D5-24, D6-6, D6-13, D6-19, D6-25, D7-27, and a combinationthereof.

In one embodiment, the human J_(H) gene segment is selected from thegroup consisting of J_(H)1, J_(H)2, J_(H)3, J_(H)4, J_(H)5, J_(H)6, anda combination thereof.

In one embodiment, the added or substituted histidine codon is presentin the unrearranged heavy chain variable region nucleotide sequenceencoding an N-terminal region, a loop 4 region, a CDR1, a CDR2, a CDR3,or a combination thereof.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprises 2 or more, 3 or more, 4 or more, 5 ormore, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 ormore, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 ormore, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 ormore, 24 or more, or 25 or more, 26 or more, 27 or more, 28 or more, 29or more, 30 or more, 31 or more, 32 or more, 33 or more, 34 or more 35or more, 36 or more, 37 or more, 38 or more, 39 or more, 40 or more, 41or more, 42 or more, 43 or more, 44 or more, 45 or more, 46 or more, 47or more, 48 or more, 49 or more, 50 or more, 51 or more, 52 or more, 53or more, 54 or more, 55 or more, 56 or more, 57 or more, 58 or more, 59or more, 60 or more, or 61 or more of histidine codons.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprising the inverted human D gene segment isoperably linked to a human or non-human heavy chain constant regionnucleotide sequence that encodes an immunoglobulin isotype selected fromIgM, IgD, IgG, IgE, and IgA.

In one embodiment, the human unrearranged immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human ornon-human heavy chain constant region nucleotide sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In oneembodiment, the heavy chain constant region nucleotide sequencecomprises a C_(H)1, a hinge, a C_(H)2, and a C_(H)3(C_(H)1-hinge-C_(H)2-C_(H)3).

In one embodiment, a heavy chain constant region nucleotide sequence ispresent at an endogenous locus (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the heavy chain constant region nucleotide sequencecomprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a modification at position 250 (e.g., E or Q); 250 and 428(e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256(e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433(e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591(e.g., V259I), and a 308F (e.g., V308F) modification; a 433K (e.g.,H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g.,252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g.,T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or308P), wherein the modification increases the affinity of the heavychain constant region amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 252 and 257,wherein the modification increases the affinity of the human C_(H)2amino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 307 and 311,wherein the modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)3 amino acid sequence, wherein the C_(H)3 aminoacid sequence comprises at least one modification between amino acidresidues at positions 433 and 436, wherein the modification increasesthe affinity of the C_(H)3 amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,N434S, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,V259I, V308F, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising an N434A mutation.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M252Y,S254T, T256E, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of T250Q,M248L, or both.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of H433K,N434Y, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises: (1) a first allele, wherein the unrearranged humanimmunoglobulin heavy chain variable region nucleotide sequence asdescribed herein is operably linked to a first heavy chain constantregion nucleotide sequence encoding a first CH₃ amino acid sequence of ahuman IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and(2) a second allele, wherein the unrearranged human immunoglobulin heavychain variable region nucleotide sequence as described herein isoperably linked to a second heavy chain constant region nucleotidesequence encoding a second C_(H)3 amino acid sequence of the human IgGselected from IgG1, IgG2, IgG4, and a combination thereof, and whereinthe second CH₃ amino acid sequence comprises a modification that reducesor eliminates binding for the second CH₃ amino acid sequence to ProteinA (see, for example, US 2010/0331527A1, incorporated by reference hereinin its entirety).

In one embodiment, the second CH₃ amino acid sequence comprises an H95Rmodification (by IMGT exon numbering; H435R by EU numbering). In oneembodiment the second CH₃ amino acid sequence further comprises an Y96Fmodification (by IMGT exon numbering; H436F by EU). In anotherembodiment, the second CH₃ amino acid sequence comprises both an H95Rmodification (by IMGT exon numbering; H435R by EU numbering) and an Y96Fmodification (by IMGT exon numbering; H436F by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG1 and further comprises a mutation selected from the groupconsisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L38M,N384S, K392N, V397M, and V422I by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG2 and further comprises a mutation selected from the groupconsisting of N44S, K52N, and V82I (IMGT: N384S, K392N, and V422I byEU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG4 and further comprises a mutation selected from the groupconsisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT: Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

In one embodiment, the heavy chain constant region amino acid sequenceis a non-human constant region amino acid sequence, and the heavy chainconstant region amino acid sequence comprises one or more of any of thetypes of modifications described above.

In one embodiment, the heavy chain constant region nucleotide sequenceis a human heavy chain constant region amino acid sequence, and thehuman heavy chain constant region amino acid sequence comprises one ormore of any of the types of modifications described above.

In one embodiment, all or substantially all endogenous V_(H), D, andJ_(H) gene segments are deleted from an immunoglobulin heavy chain locusor rendered non-functional (e.g., via insertion of a nucleotide sequence(e.g., an exogenous nucleotide sequence) in the immunoglobulin locus orvia non-functional rearrangement, or inversion, of the endogenous V_(H),D, J_(H) segments). In one embodiment, e.g., about 80% or more, about85% or more, about 90% or more, about 95% or more, about 96% or more,about 97% or more, about 98% or more, or about 99% or more of allendogenous V_(H), D, or J_(H) gene segments are deleted or renderednon-functional. In one embodiment, e.g., at least 95%, 96%, 97%, 98%, or99% of endogenous functional V, D, or J gene segments are deleted orrendered non-functional.

In one embodiment, the genetically modified immunoglobulin heavy chainlocus comprises a modification that deletes or renders, all orsubstantially all, non-functional endogenous V_(H), D, and J_(H) genesegments; and the genetically modified locus comprises an unrearrangedheavy chain variable region nucleotide sequence comprising one or morehuman V_(H), D, and/or J_(H) gene segments having one or more histidinecodons, wherein the unrearranged heavy chain variable region nucleotidesequence is present at an endogenous location (i.e., where thenucleotide sequence is located in a wild-type non-human animal) orpresent ectopically (e.g., at a locus different from the endogenousimmunoglobulin chain locus in its genome, or within its endogenouslocus, e.g., within an immunoglobulin variable locus, wherein theendogenous locus is placed or moved to a different location in thegenome).

In one embodiment, the genetically modified immunoglobulin locuscomprises an endogenous Adam6a gene, Adam6b gene, or both, and thegenetic modification does not affect the expression and/or function ofthe endogenous Adam6a gene, Adam6b gene, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises an ectopically present Adam6a gene, Adam6b gene, or both. Inone embodiment, the Adam6a gene is a non-human Adam6a gene. In oneembodiment, the Adam6a gene is a human Adam6a gene. In one embodiment,the Adam6b gene is a non-human Adam6b gene. In one embodiment, theAdam6b gene is a human Adam6b gene.

In one embodiment, the genetically modified immunoglobulin locus furthercomprises a humanized, unrearranged λ and/or κ light chain variable genesequence. In one embodiment, the humanized, unrearranged λ and/or κlight chain variable gene sequence is operably linked to animmunoglobulin light chain constant region nucleotide sequence selectedfrom a λ light chain constant region nucleotide sequence and a κ lightchain constant region nucleotide sequence. In one embodiment, thehumanized, unrearranged λ light chain variable region nucleotidesequence is operably linked to a λ light chain constant regionnucleotide sequence. In one embodiment, the λ light chain constantregion nucleotide sequence is a mouse, rat, or human sequence. In oneembodiment, the humanized, unrearranged κ light chain variable regionnucleotide sequence is operably linked to a κ light chain constantregion nucleotide sequence. In one embodiment, the κ light chainconstant region nucleotide sequence is a mouse, rat, or human sequence.

In one embodiment, the genetically modified immunoglobulin locuscomprises an unrearranged light chain variable gene sequence thatcontains at least one modification that introduces at least onehistidine codon in at least one reading frame encoding a light chainvariable domain. In one embodiment, the genetically modifiedimmunoglobulin locus comprises a rearranged (e.g., a rearranged λ or κV/J sequence) sequence that comprises one, two, three, or four codonsfor histidine in a light chain CDR. In one embodiment, the CDR is aselected from a CDR1, CDR2, CDR3, and a combination thereof. In oneembodiment, the unrearranged or rearranged light chain variable regionnucleotide sequence is an unrearranged or rearranged human λ or κ lightchain variable region nucleotide sequence. In one embodiment, theunrearranged or rearranged human λ or κ light chain variable regionnucleotide sequence is present at an endogenous mouse immunoglobulinlight chain locus. In one embodiment, the mouse immunoglobulin lightchain locus is a mouse κ locus. In one embodiment, the mouseimmunoglobulin light chain locus is a mouse λ locus.

In one embodiment, the genetically modified immunoglobulin locus asdescribed herein is present in an immunoglobulin heavy chain locus of amouse. In one embodiment, the genetically modified immunoglobulin locusis present in a humanized immunoglobulin heavy chain locus in aVELOCIMMUNE® mouse.

In one embodiment, the non-human animal is heterozygous for thegenetically modified immunoglobulin heavy chain locus, and the non-humananimal is capable of expressing a human immunoglobulin heavy chainvariable domain comprising at least one histidine residue derivedpredominantly from the genetically modified immunoglobulin heavy chainlocus as described herein.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein exhibits a weaker antigen bindingat an acidic environment (e.g., at a pH of about 5.5 to about 6.0) thana corresponding wild-type heavy chain variable domain without thegenetic modification.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification.

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises a B cell population that, upon stimulationwith an antigen of interest, is capable of producing antigen-bindingproteins, e.g., antibodies, comprising a heavy chain variable domaincomprising one or more histidine residues. The antigen-binding proteinsas described herein when administered into a subject, exhibits anincreased serum half-life over a corresponding wild-type antigen-bindingprotein, which possesses a similar or sufficiently similar amino acidsequence that encodes the heavy chain variable domain but does notcomprise a histidine residue in the heavy chain variable domain. In someembodiments, the antigen-binding protein described herein exhibits anincreased serum half-life that is at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold higher than the corresponding wild-type antigen-binding protein,which possesses a similar or sufficiently similar amino acid sequencethat encodes the heavy chain variable domain but does not comprise ahistidine residue in the heavy chain variable domain.

In one aspect, a non-human animal comprising a genetically modifiedimmunoglobulin locus is provided, wherein the genetically modifiedimmunoglobulin locus comprises an unrearranged human heavy chainvariable region nucleotide sequence, and wherein the human unrearrangedheavy chain variable region nucleotide sequence comprises a substitutionof at least one endogenous non-histidine codon with a histidine codon.

In one embodiment, the non-human animal is a mammal, including a rodent,e.g., a mouse, a rat, or a hamster.

In one embodiment, 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 ormore, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 ormore, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 ormore, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 ormore, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 ormore, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 ormore, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, 48 ormore, 49 or more, 50 or more, 51 or more, 52 or more, 53 or more, 54 ormore, 55 or more, 56 or more, 57 or more, 58 or more, 59 or more, 60 ormore, or 61 or more of the endogenous non-histidine codons are replacedwith histidine codons.

In one embodiment, the endogenous non-histone codon encodes the aminoacid selected from Y, N, D, Q, S, W, and R.

In one embodiment, the substituted histidine codon is present in anunrearranged heavy chain variable region nucleotide sequence thatencodes an immunoglobulin variable domain selected from an N-terminalregion, a loop 4 region, a CDR1, a CDR2, a CDR3, a combination thereof.

In one embodiment, the substituted histidine codon is present in anunrearranged heavy chain variable region nucleotide sequence thatencodes a complementary determining region (CDR) selected from a CDR1, aCDR2, a CDR3, and a combination thereof.

In one embodiment, the substituted histidine codon is present in anunrearranged heavy chain variable region nucleotide sequence thatencodes a frame region (FR) selected from FR1, FR2, FR3, FR4, and acombination thereof.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprises a genetically modified human V_(H) genesegment, wherein one or more endogenous non-histidine codon in at leastone reading frame of the human V_(H) gene segment has been replaced witha histidine codon.

In one embodiment, the human unrearranged heavy chain variable regionnucleotide sequence comprises a modification that replaces at least oneendogenous non-histidine codon of a human V_(H) gene segment with ahistidine codon, wherein the human V_(H) gene segment is selected fromthe group consisting of V_(H)1-2, V_(H)1-3, V_(H)1-8, V_(H)1-18,V_(H)1-24, V_(H)1-45, V_(H)1-46, V_(H)1-58, V_(H)1-69, V_(H)2-5,V_(H)2-26, V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11, V_(H)3-13,V_(H)3-15, V_(H)3-16, V_(H)3-20, V_(H)3-21, V_(H)3-23, V_(H)3-30,V_(H)3-30-3, V_(H)3-30-5, V_(H)3-33, V_(H)3-35, V_(H)3-38, V_(H)3-43,V_(H)3-48, V_(H)3-49, V_(H)3-53, V_(H)3-64, V_(H)3-66, V_(H)3-72,V_(H)3-73, V_(H)3-74, V_(H)4-4, V_(H)4-28, V_(H)4-30-1, V_(H)4-30-2,V_(H)4-30-4, V_(H)4-31, V_(H)4-34, V_(H)4-39, V_(H)4-59, V_(H)4-61,V_(H)5-51, V_(H)6-1, V_(H)7-4-1, V_(H)7-81, and a combination thereof.

In one embodiment, the human unrearranged heavy chain variable regionnucleotide sequence comprises a genetically modified human J_(H) genesegment, wherein one or more endogenous non-histidine codon in at leastone reading frame of the human J_(H) gene segment has been replaced witha histidine codon.

In one embodiment, the human unrearranged heavy chain variable regionnucleotide sequence comprises a modification that replaces at least oneendogenous non-histidine codon of a human J_(H) segment with a histidinecodon, wherein the human J_(H) gene segment is selected from the groupconsisting of J_(H)1, J_(H)2, J_(H)3, J_(H)4, J_(H)5, J_(H)6, and acombination thereof.

In one embodiment, the substituted histidine codon is present in a heavychain variable region nucleotide sequence that encodes part of a CDR3.In one embodiment, the part of CDR3 comprises an amino acid sequencederived from a reading frame of a genetically modified human D genesegment comprising a modification that replaces at least one endogenousnon-histidine codon in the reading frame with a histidine codon.

In one embodiment, the endogenous non-histidine codon that issubstituted with a histidine codon encodes the amino acid selected fromY, N, D, Q, S, W, and R.

In one embodiment, the substituted histidine codon is present in atleast one reading frame of the human D gene segment that is mostfrequently observed in VELOCIMMUNE® humanized immunoglobulin mice.

In one embodiment, the reading frame of the genetically modified human Dgene segment that encodes part of CDR3 is selected from a hydrophobicframe, a stop frame, and a hydrophilic frame.

In one embodiment, the reading frame is a hydrophobic frame of a human Dgene segment.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D1-1 (GTTGT; SEQ ID NO: 88), D1-7(GITGT; SEQ ID NO: 89), D1-20 (GITGT; SEQ ID NO: 89), and D1-26 (GIVGAT;SEQ ID NO:90), and the human D gene segment further comprises amodification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D2-2 (DIVVVPAAI; SEQ ID NO:92),D2-8 (DIVLMVYAI; SEQ ID NO: 94), D2-15 (DIVVVVAAT; SEQ ID NO:95), andD2-21 (HIVVVTAI; SEQ ID NO: 97), and the human D gene segment furthercomprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D3-3 (ITIFGVVII; SEQ ID NO:98),D3-9 (ITIF*LVII; SEQ ID NO:99, SEQ ID NO:100), D3-10 (ITMVRGVII; SEQ IDNO:101), D3-16 (IMITFGGVIVI; SEQ ID NO:102), and D3-22 (ITMIVVVIT; SEQID NO:103), and the human D gene segment further comprises amodification that replaces at least one endogenous codon in thenucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D4-4 (TTVT; SEQ ID NO:105), D4-11(TTVT; SEQ ID NO:105), D4-17 (TTVT; SEQ ID NO:105), D4-23 (TTWT; SEQ IDNO: 106) and the human D gene segment further comprises a modificationthat replaces at least one endogenous non-histidine codon in thenucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D5-5 (VDTAMV; SEQ ID NO: 107),D5-12 (VDIVATI; SEQ ID NO:108), D5-18 (VDTAMV; SEQ ID NO:107), and D5-24(VEMATI; SEQ ID NO:109), and the human D gene segment further comprisesa modification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D6-6 (SIAAR; SEQ ID NO:111), D6-13(GIAAAG; SEQ ID NO:113), and D6-19 (GIAVAG; SEQ ID NO:115), and thehuman D gene segment further comprises a modification that replaces atleast one endogenous non-histidine codon in the nucleotide sequence witha histidine codon.

In one embodiment, the hydrophobic frame comprises a nucleotide sequencethat encodes human D7-27 (LTG), and the human D gene segment furthercomprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the reading frame is a stop reading frame of a humanD gene segment.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D1-1 (VQLER; SEQ ID NO:8),D1-7(V*LEL), D1-20(V*LER), D1-26 (V*WELL; SEQ ID NO:12), and the human Dgene segment further comprises a modification that replaces at least oneendogenous non-histidine codon in the nucleotide sequence with ahistidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D2-2 (RIL**YQLLY; SEQ ID NO:14),D2-8 (RILY*WCMLY; SEQ ID NO:16 and SEQ ID NO: 17), D2-15 (RIL*WW*LLL),and D2-21 (SILWW*LLF; SEQ ID NO:19), and the human D gene segmentfurther comprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D3-3 (VLRFLEWLLY; SEQ ID NO:21),D3-9 (VLRYFDWLL*; SEQ ID NO:23), D3-10 (VLLWFGELL*; SEQ ID NO:25), D3-16(VL*LRLGELSLY; SEQ ID NO:27), and D3-22 (VLL***WLLL; SEQ ID NO:29), andthe human D gene segment comprises a modification that replaces at leastone endogenous non-histidine codon in the nucleotide sequence with ahistidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D4-4 (*LQ*L), D4-11 (*LQ*L), D4-17(*LR*L), and D4-23 (*LRW*L), and the human D gene segment comprises amodification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D5-5 (WIQLWL; SEQ ID NO:35); D5-12(WI*WLRL; SEQ ID NO:37), D5-18 (WIQLWL; SEQ ID NO:35), and D5-24(*RWLQL; SEQ ID NO:39), and the human D gene segment comprises amodification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D6-6 (V*QLV), D6-13 (V*QQLV; SEQID NO:41), and D6-19 (V*QWLV; SEQ ID NO:43), and the human D genesegment further comprises a modification that replaces at least oneendogenous non-histidine codon in the nucleotide sequence with ahistidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes D7-27 (*LG), and the humanD gene segment further comprises a modification that replaces at leastone endogenous codon of the human D gene segment in the nucleotidesequence with a histidine codon.

In one embodiment, the reading frame is a hydrophilic frame of a human Dgene segment.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D1-1 (YNWND; SEQ ID NO: 45), D1-7(YNWNY; SEQ ID NO: 47), D1-20 (YNWND; SEQ ID NO: 45), and D1-26 (YSGSYY;SEQ ID NO:49), and the human D gene segment further comprises amodification that replaces at least one endogenous codon in thenucleotide sequence with a histidine codon. In one embodiment, thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 46, SEQID NO: 48, SEQ ID NO: 50, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D2-2 (GYCSSTSCYT; SEQ ID NO:51),D2-8 (GYCTNGVCYT; SEQ ID NO: 53), D2-15 (GYCSGGSCYS; SEQ ID NO:55), andD2-21 (AYCGGDCYS; SEQ ID NO:57), and the human D gene segment furthercomprises a modification that replaces at least one endogenous codon inthe nucleotide sequence with a histidine codon. In one embodiment, thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 52, SEQID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D3-3 (YYDFWSGYYT; SEQ ID NO:59),D3-9 (YYDILTGYYN; SEQ ID NO:61), D3-10 (YYYGSGSYYN; SEQ ID NO:63), D3-16(YYDYVWGSYRYT; SEQ ID NO:65), and D3-22 (YYYDSSGYYY; SEQ ID NO:67), andthe human D gene segment further comprises a modification that replacesat least one endogenous codon in the nucleotide sequence with ahistidine codon. In one embodiment, the hydrophilic frame comprises anucleotide sequence encodes the amino acid sequence selected from thegroup consisting of SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ IDNO: 66, SEQ ID NO: 68, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D4-4 (DYSNY; SEQ ID NO:69), D4-11(DYSNY; SEQ ID NO:69), D4-17 (DYGDY; SEQ ID NO:71), and D4-23 (DYGGNS;SEQ ID NO:73), and the human D gene segment comprises a modificationthat replaces at least one endogenous codon in the nucleotide sequencewith a histidine codon. In one embodiment, the hydrophilic framecomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 72, SEQID NO: 74, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D5-5 (GYSYGY; SEQ ID NO:75), D5-12(GYSGYDY; SEQ ID NO:77), D5-18 (GYSYGY; SEQ ID NO:75), and D5-24(RDGYNY; SEQ ID NO:79), and the human D gene segment further comprises amodification that replaces at least one endogenous codon in thenucleotide sequence with a histidine codon. In one embodiment, thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 76, SEQID NO: 78, SEQ ID NO: 80, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D6-6 (EYSSSS; SEQ ID NO: 81),D6-13 (GYSSSWY; SEQ ID NO:83), and D6-19 (GYSSGWY; SEQ ID NO:85), andthe human D gene segment further comprises a modification that replacesat least one endogenous codon in the nucleotide sequence with ahistidine codon. In one embodiment, the hydrophilic frame comprises anucleotide sequence that encodes the amino acid sequence selected fromthe group consisting of SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQID NO: 76, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes D7-27 (NWG), and the humanD gene segment further comprises a modification that replaces at leastone endogenous codon in the nucleotide sequence a histidine codon.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO:68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ IDNO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, anda combination thereof.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprising the inverted human D gene segment isoperably linked to a human or non-human heavy chain constant regionnucleotide sequence that encodes an immunoglobulin isotype selected fromIgM, IgD, IgG, IgE, and IgA.

In one embodiment, the human unrearranged immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human ornon-human heavy chain constant region nucleotide sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In oneembodiment, the heavy chain constant region nucleotide sequencecomprises a C_(H)1, a hinge, a C_(H)2, and a C_(H)3(C_(H)1-hinge-C_(H)2-C_(H)3).

In one embodiment, a heavy chain constant region nucleotide sequence ispresent at an endogenous locus (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the heavy chain constant region nucleotide sequencecomprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a modification at position 250 (e.g., E or Q); 250 and 428(e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256(e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433(e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591(e.g., V259I), and a 308F (e.g., V308F) modification; a 433K (e.g.,H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g.,252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g.,T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or308P), wherein the modification increases the affinity of the heavychain constant region amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 252 and 257,wherein the modification increases the affinity of the human C_(H)2amino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 307 and 311,wherein the modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)3 amino acid sequence, wherein the C_(H)3 aminoacid sequence comprises at least one modification between amino acidresidues at positions 433 and 436, wherein the modification increasesthe affinity of the C_(H)3 amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,N434S, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,V259I, V308F, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising an N434A mutation.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M252Y,S254T, T256E, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of T250Q,M248L, or both.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of H433K,N434Y, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises: (1) a first allele, wherein the unrearranged humanimmunoglobulin heavy chain variable region nucleotide sequence asdescribed herein is operably linked to a first heavy chain constantregion nucleotide sequence encoding a first CH₃ amino acid sequence of ahuman IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and(2) a second allele, wherein the unrearranged human immunoglobulin heavychain variable region nucleotide sequence as described herein isoperably linked to a second heavy chain constant region nucleotidesequence encoding a second C_(H)3 amino acid sequence of the human IgGselected from IgG1, IgG2, IgG4, and a combination thereof, and whereinthe second CH₃ amino acid sequence comprises a modification that reducesor eliminates binding for the second CH₃ amino acid sequence to ProteinA (see, for example, US 2010/0331527A1, incorporated by reference hereinin its entirety).

In one embodiment, the second CH₃ amino acid sequence comprises an H95Rmodification (by IMGT exon numbering; H435R by EU numbering). In oneembodiment the second CH₃ amino acid sequence further comprises an Y96Fmodification (by IMGT exon numbering; H436F by EU). In anotherembodiment, the second CH₃ amino acid sequence comprises both an H95Rmodification (by IMGT exon numbering; H435R by EU numbering) and an Y96Fmodification (by IMGT exon numbering; H436F by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG1 and further comprises a mutation selected from the groupconsisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L38M,N384S, K392N, V397M, and V422I by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG2 and further comprises a mutation selected from the groupconsisting of N44S, K52N, and V82I (IMGT: N384S, K392N, and V422I byEU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG4 and further comprises a mutation selected from the groupconsisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT: Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

In one embodiment, the heavy chain constant region amino acid sequenceis a non-human constant region amino acid sequence, and the heavy chainconstant region amino acid sequence comprises one or more of any of thetypes of modifications described above.

In one embodiment, the heavy chain constant region nucleotide sequenceis a human heavy chain constant region amino acid sequence, and thehuman heavy chain constant region amino acid sequence comprises one ormore of any of the types of modifications described above.

In one embodiment, all or substantially all endogenous V_(H), D, andJ_(H) gene segments are deleted from an immunoglobulin heavy chain locusor rendered non-functional (e.g., via insertion of a nucleotide sequence(e.g., an exogenous nucleotide sequence) in the immunoglobulin locus orvia non-functional rearrangement, or inversion, of the endogenous V_(H),D, J_(H) segments). In one embodiment, e.g., about 80% or more, about85% or more, about 90% or more, about 95% or more, about 96% or more,about 97% or more, about 98% or more, or about 99% or more of allendogenous V_(H), D, or J_(H) gene segments are deleted or renderednon-functional. In one embodiment, e.g., at least 95%, 96%, 97%, 98%, or99% of endogenous functional V, D, or J gene segments are deleted orrendered non-functional.

In one embodiment, the genetically modified locus comprises amodification that deletes or renders non-functional all or substantiallyall endogenous V_(H), D, and J_(H) gene segments; and the genomic locuscomprises the genetically modified, unrearranged human heavy chainvariable region nucleotide sequence comprising a substitution of atleast one endogenous non-histidine codon with a histidine codon in atleast one reading frame. In one embodiment, the genetically modified,unrearranged immunoglobulin heavy chain variable gene sequence ispresent at an endogenous location (i.e., where the nucleotide sequenceis located in a wild-type non-human animal) or present ectopically(e.g., at a locus different from the endogenous immunoglobulin chainlocus in its genome or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the genetically modified locus comprises anendogenous Adam6a gene, Adam6b gene, or both, and the geneticmodification does not affect the expression and/or function of theendogenous Adam6a gene, Adam6b gene, or both.

In one embodiment, the genetically modified locus comprises anectopically present Adam6a gene, Adam6b gene, or both. In oneembodiment, the Adam6a gene is a non-human Adam6a gene. In oneembodiment, the Adam6a gene is a mouse Adam6a gene. In one embodiment,the Adam6a gene is a human Adam6a gene. In one embodiment, the Adam6bgene is a non-human Adam6b gene. In one embodiment, the Adam6b gene is amouse Adam6b gene. In one embodiment, the Adam6b gene is a human Adam6bgene.

In one embodiment, the genetically modified immunoglobulin locus furthercomprises a humanized, unrearranged λ and/or κ light chain variable genesequence. In one embodiment, the humanized, unrearranged λ and/or κlight chain variable gene sequence is operably linked to animmunoglobulin light chain constant region nucleotide sequence selectedfrom a λ light chain constant region nucleotide sequence and a κ lightchain constant region nucleotide sequence. In one embodiment, thehumanized, unrearranged λ light chain variable region nucleotidesequence is operably linked to a λ light chain constant regionnucleotide sequence. In one embodiment, the λ light chain constantregion nucleotide sequence is a mouse, rat, or human sequence. In oneembodiment, the humanized, unrearranged κ light chain variable regionnucleotide sequence is operably linked to a κ light chain constantregion nucleotide sequence. In one embodiment, the κ light chainconstant region nucleotide sequence is a mouse, rat, or human sequence.

In one embodiment, the genetically modified immunoglobulin locuscomprises an unrearranged light chain variable gene sequence thatcontains at least one modification that introduces at least onehistidine codon in at least one reading frame encoding a light chainvariable domain. In one embodiment, the genetically modifiedimmunoglobulin locus comprises a rearranged (e.g., rearranged λ or κ V/Jsequence) sequence that comprises one, two, three, or four codons forhistidine in a light chain CDR. In one embodiment, the CDR is a selectedfrom a CDR1, CDR2, CDR3, and a combination thereof. In one embodiment,the unrearranged or rearranged light chain variable region nucleotidesequence is an unrearranged or rearranged human λ or κ light chainvariable region nucleotide sequence. In one embodiment, the unrearrangedor rearranged human λ or κ light chain variable region nucleotidesequence is present at an endogenous mouse immunoglobulin light chainlocus. In one embodiment, the mouse immunoglobulin light chain locus isa mouse κ locus. In one embodiment the mouse immunoglobulin light chainlocus is a mouse λ locus.

In one embodiment, the genetically modified immunoglobulin locus asdescribed herein is present in an immunoglobulin heavy chain locus of amouse. In one embodiment, the genetically modified immunoglobulin locusis present in a humanized immunoglobulin heavy chain locus in aVELOCIMMUNE® mouse.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein exhibits a weaker antigen bindingat an acidic environment (e.g., at a pH of about 5.5 to about 6.0) thana corresponding wild-type heavy chain variable domain without thegenetic modification.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification.

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises a B cell population that, upon stimulationwith an antigen of interest, is capable of producing antigen-bindingproteins, e.g., antibodies, comprising a heavy chain variable domaincomprising one or more histidine residues. The antigen-binding proteinsas described herein when administered into a subject, exhibits anincreased serum half-life over a corresponding wild-type antigen-bindingprotein, which possesses a similar or sufficiently similar amino acidsequence that encodes the heavy chain variable domain but does notcomprise a histidine residue in the heavy chain variable domain. In someembodiments, the antigen-binding protein described herein exhibits anincreased serum half-life that is at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold higher than the corresponding wild-type antigen-binding protein,which possesses a similar or sufficiently similar amino acid sequencethat encodes the heavy chain variable domain but does not comprise ahistidine residue in the heavy chain variable domain.

In one embodiment, the non-human animal is heterozygous for thegenetically modified immunoglobulin heavy chain locus, and the non-humananimal is capable of expressing the human immunoglobulin heavy chainvariable domain comprising at least one histidine residue derivedpredominantly from the genetically modified immunoglobulin heavy chainlocus as described herein.

In one aspect, a non-human animal comprising a genetically modifiedimmunoglobulin locus comprising a human V_(H), D, and J_(H) gene segmentis provided, wherein at least one of the human D gene segment has beeninverted 5′ to 3′ with respect to a corresponding wild-type sequence,and wherein at least one reading frame of the inverted human D genesegment comprises a histidine codon.

In one embodiment, the non-human animal is a mammal, including a rodent,e.g., a mouse, a rat, or a hamster

In one embodiment, the genetically modified immunoglobulin locus ispresent in a germline genome.

In one embodiment, wherein the reading frame of the inverted human Dgene segment comprises one or more, 2 or more, 3 or more, 4 or more, 5or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 ormore, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 ormore, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 ormore, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 ormore, 30 or more, 31 or more, 32 or more, 33 or more, or 34 or more ofhistidine codons.

In one embodiment, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least nine, atleast ten, at least eleven, at least twelve, at least thirteen, at leastfourteen, at least fifteen, at least sixteen, at least seventeen, atleast eighteen, at least nineteen, at least twenty, at least twenty one,at least twenty two, at least twenty three, at least twenty four, or allor substantially all of functional human D gene segments have invertedorientation with respect to corresponding wild type sequences.

In one embodiment, all or substantially all of endogenous immunoglobulinV_(H), D, J_(H) gene segments are deleted from the immunoglobulin heavychain locus or rendered non-functional (e.g., via insertion of anucleotide sequence, e.g., exogenous nucleotide sequence, in theimmunoglobulin locus or via non-functional rearrangement or inversion ofall, or substantially all, endogenous immunoglobulin V_(H), D, J_(H)segments), and the genetically modified immunoglobulin locus comprises ahuman V_(H), D, and J_(H) gene segments, wherein at least one of thehuman D gene segment is present in an inverted orientation with respectto corresponding wild type sequences, and wherein at least one readingframe of the inverted human D gene segment comprises at least onehistidine codon.

In one embodiment, the inverted human D gene segment is operably linkedto a human V_(H) gene segment, and/or human J_(H) gene segment

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence isselected from the group consisting of D1-1, D1-7, D1-20, D1-26, D2-2,D2-8, D2-15, D2-21, D3-3, D3-9, D3-10, D3-16, D3-22, D4-4, D4-11, D4-17,D4-23, D5-5, D5-12, D5-18, D5-24, D6-6, D6-13, D6-19, D7-27, and acombination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD1 gene segment selected from the group consisting of D1-1, D1-7, D1-20,D1-26, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequences isa D2 gene segment selected from the group consisting of D2-2, D2-8,D2-15, D2-21, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD3 gene segment selected from the group consisting of D3-3, D3-9, D3-10,D3-16, D3-22, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD4 gene segment selected from the group consisting of D4-4, D4-11,D4-17, D4-23, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD5 gene segment selected from the group consisting of D5-5, D5-12,D5-18, D5-24, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD6 gene segment selected from the group consisting of D6-6, D6-13,D6-19, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence isD7-27.

In one embodiment, the reading frame of the human D gene segment isselected from a stop reading frame, a hydrophilic reading frame, ahydrophobic reading frame, and a combination thereof, wherein at leastone reading frame of the inverted human D gene segment comprises ahistidine codon.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprising the inverted human D gene segment isoperably linked to a human or non-human heavy chain constant regionnucleotide sequence that encodes an immunoglobulin isotype selected fromIgM, IgD, IgG, IgE, and IgA.

In one embodiment, the human unrearranged immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human ornon-human heavy chain constant region nucleotide sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In oneembodiment, the heavy chain constant region nucleotide sequencecomprises a C_(H)1, a hinge, a C_(H)2, and a C_(H)3(C_(H)1-hinge-C_(H)2-C_(H)3).

In one embodiment, a heavy chain constant region nucleotide sequence ispresent at an endogenous locus (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the heavy chain constant region nucleotide sequencecomprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a modification at position 250 (e.g., E or Q); 250 and 428(e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256(e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433(e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591(e.g., V259I), and a 308F (e.g., V308F) modification; a 433K (e.g.,H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g.,252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g.,T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or308P), wherein the modification increases the affinity of the heavychain constant region amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 252 and 257,wherein the modification increases the affinity of the human C_(H)2amino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 307 and 311,wherein the modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)3 amino acid sequence, wherein the C_(H)3 aminoacid sequence comprises at least one modification between amino acidresidues at positions 433 and 436, wherein the modification increasesthe affinity of the C_(H)3 amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,N434S, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,V259I, V308F, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising an N434A mutation.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M252Y,S254T, T256E, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of T250Q,M248L, or both.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of H433K,N434Y, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises: (1) a first allele, wherein the unrearranged humanimmunoglobulin heavy chain variable region nucleotide sequence asdescribed herein is operably linked to a first heavy chain constantregion nucleotide sequence encoding a first CH₃ amino acid sequence of ahuman IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and(2) a second allele, wherein the unrearranged human immunoglobulin heavychain variable region nucleotide sequence as described herein isoperably linked to a second heavy chain constant region nucleotidesequence encoding a second C_(H)3 amino acid sequence of the human IgGselected from IgG1, IgG2, IgG4, and a combination thereof, and whereinthe second CH₃ amino acid sequence comprises a modification that reducesor eliminates binding for the second CH₃ amino acid sequence to ProteinA (see, for example, US 2010/0331527A1, incorporated by reference hereinin its entirety).

In one embodiment, the second CH₃ amino acid sequence comprises an H95Rmodification (by IMGT exon numbering; H435R by EU numbering). In oneembodiment the second CH₃ amino acid sequence further comprises an Y96Fmodification (by IMGT exon numbering; H436F by EU). In anotherembodiment, the second CH₃ amino acid sequence comprises both an H95Rmodification (by IMGT exon numbering; H435R by EU numbering) and an Y96Fmodification (by IMGT exon numbering; H436F by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG1 and further comprises a mutation selected from the groupconsisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L38M,N384S, K392N, V397M, and V422I by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG2 and further comprises a mutation selected from the groupconsisting of N44S, K52N, and V82I (IMGT: N384S, K392N, and V422I byEU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG4 and further comprises a mutation selected from the groupconsisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT: Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

In one embodiment, the heavy chain constant region amino acid sequenceis a non-human constant region amino acid sequence, and the heavy chainconstant region amino acid sequence comprises one or more of any of thetypes of modifications described above.

In one embodiment, the heavy chain constant region nucleotide sequenceis a human heavy chain constant region amino acid sequence, and thehuman heavy chain constant region amino acid sequence comprises one ormore of any of the types of modifications described above.

In one embodiment, all or substantially all endogenous V_(H), D, andJ_(H) gene segments are deleted from an immunoglobulin heavy chain locusor rendered non-functional (e.g., via insertion of a nucleotide sequence(e.g., an exogenous nucleotide sequence) in the immunoglobulin locus orvia non-functional rearrangement, or inversion, of the endogenous V_(H),D, J_(H) segments). In one embodiment, e.g., about 80% or more, about85% or more, about 90% or more, about 95% or more, about 96% or more,about 97% or more, about 98% or more, or about 99% or more of allendogenous V_(H), D, or J_(H) gene segments are deleted or renderednon-functional. In one embodiment, e.g., at least 95%, 96%, 97%, 98%, or99% of endogenous functional V, D, or J gene segments are deleted orrendered non-functional.

In one embodiment, the genetically modified immunoglobulin heavy chainlocus comprises a modification that deletes or renders, all orsubstantially all, non-functional endogenous V_(H), D, and J_(H) genesegments; and the genetically modified locus comprises an unrearrangedheavy chain variable region nucleotide sequence comprising at least oneinverted human D gene segment as described herein wherein theunrearranged heavy chain variable region nucleotide sequence is presentat an endogenous location (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the genetically modified immunoglobulin locuscomprises an endogenous Adam6a gene, Adam6b gene, or both, and thegenetic modification does not affect the expression and/or function ofthe endogenous Adam6a gene, Adam6b gene, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises an ectopically present Adam6a gene, Adam6b gene, or both. Inone embodiment, the Adam6a gene is a non-human Adam6a gene. In oneembodiment, the Adam6a gene is a mouse Adam6a gene. In one embodiment,the Adam6a gene is a human Adam6a gene. In one embodiment, the Adam6bgene is a non-human Adam6b gene. In one embodiment, the Adam6b gene is amouse Adam6b gene. In one embodiment, the Adam6b gene is a human Adam6bgene.

In one embodiment, the genetically modified immunoglobulin locus furthercomprises a humanized, unrearranged λ and/or κ light chain variable genesequence. In one embodiment, the humanized, unrearranged λ and/or κlight chain variable gene sequence is operably linked to animmunoglobulin light chain constant region nucleotide sequence selectedfrom a λ light chain constant region nucleotide sequence and a κ lightchain constant region nucleotide sequence. In one embodiment, thehumanized, unrearranged λ light chain variable region nucleotidesequence is operably linked to a λ light chain constant regionnucleotide sequence. In one embodiment, the λ light chain constantregion nucleotide sequence is a mouse, rat, or human sequence. In oneembodiment, the humanized, unrearranged κ light chain variable regionnucleotide sequence is operably linked to a κ light chain constantregion nucleotide sequence. In one embodiment, the κ light chainconstant region nucleotide sequence is a mouse, rat, or human sequence.

In one embodiment, the genetically modified immunoglobulin locuscomprises an unrearranged light chain variable gene sequence thatcontains at least one modification that introduces at least onehistidine codon in at least one reading frame encoding a light chainvariable domain. In one embodiment, the genetically modifiedimmunoglobulin locus comprises a rearranged (e.g., a rearranged λ or κV/J sequence) sequence that comprises one, two, three, or four codonsfor histidine in a light chain CDR. In one embodiment, the CDR is aselected from a CDR1, CDR2, CDR3, and a combination thereof. In oneembodiment, the unrearranged or rearranged light chain variable regionnucleotide sequence is an unrearranged or rearranged human λ or κ lightchain variable region nucleotide sequence. In one embodiment, theunrearranged or rearranged human λ or κ light chain variable regionnucleotide sequence is present at an endogenous mouse immunoglobulinlight chain locus. In one embodiment, the mouse immunoglobulin lightchain locus is a mouse κ locus. In one embodiment, the mouseimmunoglobulin light chain locus is a mouse immunoglobulin light chainlocus is a mouse λ locus.

In one embodiment, the genetically modified immunoglobulin locus asdescribed herein is present in an immunoglobulin heavy chain locus of amouse. In one embodiment, the genetically modified immunoglobulin locusis present in a humanized immunoglobulin heavy chain locus in aVELOCIMMUNE® mouse.

In one embodiment, the non-human animal is heterozygous for thegenetically modified immunoglobulin heavy chain locus, and the non-humananimal is capable of expressing the human immunoglobulin heavy chainvariable domain comprising at least one histidine residue derivedpredominantly from the genetically modified immunoglobulin heavy chainlocus as described herein.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein exhibits a weaker antigen bindingat an acidic environment (e.g., at a pH of about 5.5 to about 6.0) thana corresponding wild-type heavy chain variable domain without thegenetic modification.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification.

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises a B cell population that, upon stimulationwith an antigen of interest, is capable of producing antigen-bindingproteins, e.g., antibodies, comprising a heavy chain variable domaincomprising one or more histidine residues. The antigen-binding proteinsas described herein when administered into a subject, exhibits anincreased serum half-life over a corresponding wild-type antigen-bindingprotein, which possesses a similar or sufficiently similar amino acidsequence that encodes the heavy chain variable domain but does notcomprise a histidine residue in the heavy chain variable domain. In someembodiments, the antigen-binding protein described herein exhibits anincreased serum half-life that is at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold higher than the corresponding wild-type antigen-binding protein,which possesses a similar or sufficiently similar amino acid sequencethat encodes the heavy chain variable domain but does not comprise ahistidine residue in the heavy chain variable domain.

In one aspect, a non-human animal that is capable of expressing anantigen-binding protein with enhanced pH-dependent recyclability and/orenhanced serum half-life are provided, wherein the non-human animalcomprises in its germline genome an unrearranged human immunoglobulinheavy chain variable region nucleotide sequence, wherein theunrearranged heavy chain variable region nucleotide sequence comprisesan addition of least one histidine codon or a substitution of at leastone endogenous non-histidine codon with a histidine codon as describedherein.

In one embodiment, the antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein exhibits a weaker antigen bindingat an acidic environment (e.g., at a pH of about 5.5 to about 6.0) thana corresponding wild-type heavy chain variable domain without thegenetic modification.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, the antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification.

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises a B cell population that, upon stimulationwith an antigen of interest, is capable of producing antigen-bindingproteins, e.g., antibodies, comprising a heavy chain variable domaincomprising one or more histidine residues. The antigen-binding proteinsas described herein when administered into a subject, exhibits anincreased serum half-life over a corresponding wild-type antigen-bindingprotein, which possesses a similar or sufficiently similar amino acidsequence that encodes the heavy chain variable domain but does notcomprise a histidine residue in the heavy chain variable domain. In someembodiments, the antigen-binding protein described herein exhibits anincreased serum half-life that is at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold higher than the corresponding wild-type antigen-binding protein,which possesses a similar or sufficiently similar amino acid sequencethat encodes the heavy chain variable domain but does not comprise ahistidine residue in the heavy chain variable domain.

In one aspect, a targeting construct is provided, comprising 5′ and 3′targeting arms homologous to a genomic D region or genomic V and Jregion of a non-human animal, wherein at least one V_(H), D, or J_(H)gene segment comprises any of the modifications as described herein,e.g., an addition of at least one histidine codon, a substitution of atleast one endogenous non-histidine codon into a histidine codon, and/orinversion of at least one functional D gene segment with respect to acorresponding wild type sequence.

In one aspect, a hybridoma or quadroma is provided that is derived froma cell of any of the non-human animal as described herein. In oneembodiment, the non-human animal is a rodent, e.g., a mouse, a rat, or ahamster.

In one aspect, pluripotent, induced pluripotent, or totipotent stemcells derived form a non-human animal comprising the various genomicmodifications of the described invention are provided. In a specificembodiment, the pluripotent, induced pluripotent, or totipotent stemcells are mouse or rat embryonic stem (ES) cells. In one embodiment, thepluripotent, induced pluripotent, or totipotent stem cells have an XXkaryotype or an XY karyotype. In one embodiment, the pluripotent orinduced pluripotent stem cells are hematopoietic stem cells.

In one aspect, cells that comprise a nucleus containing a geneticmodification as described herein are also provided, e.g., a modificationintroduced into a cell by pronuclear injection. In one embodiment, thepluripotent, induced pluripotent, or totipotent stem cells comprise agenetically modified immunoglobulin genomic locus, wherein the genomiclocus comprises, from 5′ to 3′, (1) an FRT recombination site, (2) humanV_(H) gene segments, (3) a mouse adam6 gene, (4) a loxP recombinationsite, (5) histidine-substituted human D gene segments, (6) human J_(H)gene segments, followed by (7) a mouse E, (intronic enhancer), and (8) amouse IgM constant region nucleotide sequence.

In one aspect, a lymphocyte isolated from a genetically modifiednon-human animal as described herein is provided. In one embodiment, thelymphocyte is a B cell, wherein the B cell comprises an immunoglobulingenomic locus comprising an unrearranged heavy chain variable regionnucleotide sequence wherein the unrearranged heavy chain variable genesequence comprises an addition of least one histidine codon or asubstitution of at least one endogenous non-histidine codon with ahistidine codon.

In one aspect, a lymphocyte isolated from a genetically modifiednon-human animal as described herein is provided. In one embodiment, thelymphocyte is a B cell, wherein the B cell comprises an immunoglobulinlocus that comprises a human V, D, and J gene segment, wherein at leastone of the human D gene segment has been inverted 5′ to 3′ with respectto wild-type sequences, and wherein at least one reading frame of theinverted human D gene segment encodes at least one histidine residue. Inone embodiment, the B cell is capable of producing an antigen-bindingprotein comprising the genetically modified heavy chain variable domainas described herein. In one embodiment, the genetically modified heavychain variable domain as described herein is operably linked to a heavychain constant region amino acid sequence.

In one aspect, a B cell population is provided that are capable ofexpressing an antigen-binding protein wherein the antigen-bindingprotein comprises at least one histidine residue in a heavy chainvariable domain, wherein the B cell population comprises any geneticmodifications as described herein. In one embodiment, the at least onehistidine residue is present in a heavy chain CDR. In one embodiment,the CDR is a selected from a CDR1, CDR2, CDR3, and a combinationthereof. In one embodiment, the at least one histidine residue ispresent in CDR3.

In one aspect, a B cell population is provided that are capable ofexpressing an antigen-binding protein with enhanced serum half-lifeand/or enhanced pH-dependent recyclability, wherein the B cellpopulation comprises any genetic modifications as described herein.

In one aspect, a method for making a non-human animal comprising agenetically modified immunoglobulin heavy chain variable locus isprovided, comprising: (a) modifying a genome of a non-human animal todelete or render non-functional endogenous immunoglobulin heavy chain V,D, and J gene segments (e.g., via insertion of a nucleotide sequence,e.g., an exogenous nucleotide sequence, in the immunoglobulin locus orvia non-functional rearrangement or inversion of endogenous V_(H), D,J_(H) segments); and (b) placing in the genome an unrearranged heavychain variable region nucleotide sequence, wherein the unrearrangedheavy chain variable region nucleotide sequence comprises an addition ofleast one histidine codon or a substitution of at least one endogenousnon-histidine codon with a histidine codon as described herein.

In one embodiment, the non-human animal is a mammal, including a rodent,e.g., a mouse, a rat, or a hamster.

In one embodiment, 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 ormore, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 ormore, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 ormore, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 ormore, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 ormore, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 ormore, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, 48 ormore, 49 or more, 50 or more, 51 or more, 52 or more, 53 or more, 54 ormore, 55 or more, 56 or more, 57 or more, 58 or more, 59 or more, 60 ormore, or 61 or more of the endogenous non-histidine codons are replacedwith histidine codons.

In one embodiment, the endogenous non-histone codon encodes the aminoacid selected from Y, N, D, Q, S, W, and R.

In one embodiment, the added or substituted histidine codon is presentin an unrearranged heavy chain variable region nucleotide sequence thatencodes an immunoglobulin variable domain selected from an N-terminalregion, a loop 4 region, a CDR1, a CDR2, a CDR3, a combination thereof.

In one embodiment, the added substituted histidine codon histidine codonis present in an unrearranged heavy chain variable region nucleotidesequence that encodes a complementary determining region (CDR) selectedfrom a CDR1, a CDR2, a CDR3, and a combination thereof.

In one embodiment, the added or substituted histidine codon is presentin an unrearranged heavy chain variable region nucleotide sequence thatencodes a frame region (FR) selected from FR1, FR2, FR3, FR4, and acombination thereof.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprises a genetically modified human V_(H) genesegment, wherein one or more endogenous non-histidine codon in at leastone reading frame of the human V_(H) gene segment has been replaced witha histidine codon.

In one embodiment, the human unrearranged heavy chain variable regionnucleotide sequence comprises a modification that replaces at least oneendogenous non-histidine codon of a human V_(H) gene segment with ahistidine codon, wherein the human V_(H) gene segment is selected fromthe group consisting of V_(H)1-2, V_(H)1-3, V_(H)1-8, V_(H)1-18,V_(H)1-24, V_(H)1-45, V_(H)1-46, V_(H)1-58, V_(H)1-69, V_(H)2-5,V_(H)2-26, V_(H)2-70, V_(H)3-7, V_(H)3-9, V_(H)3-11, V_(H)3-13,V_(H)3-15, V_(H)3-16, V_(H)3-20, V_(H)3-21, V_(H)3-23, V_(H)3-30,V_(H)3-30-3, V_(H)3-30-5, V_(H)3-33, V_(H)3-35, V_(H)3-38, V_(H)3-43,V_(H)3-48, V_(H)3-49, V_(H)3-53, V_(H)3-64, V_(H)3-66, V_(H)3-72,V_(H)3-73, V_(H)3-74, V_(H)4-4, V_(H)4-28, V_(H)4-30-1, V_(H)4-30-2,V_(H)4-30-4, V_(H)4-31, V_(H)4-34, V_(H)4-39, V_(H)4-59, V_(H)4-61,V_(H)5-51, V_(H)6-1, V_(H)7-4-1, V_(H)7-81, and a combination thereof.

In one embodiment, the human unrearranged heavy chain variable regionnucleotide sequence comprises a genetically modified human J_(H) genesegment, wherein one or more endogenous non-histidine codon in at leastone reading frame of the human J_(H) gene segment has been replaced witha histidine codon.

In one embodiment, the human unrearranged heavy chain variable regionnucleotide sequence comprises a modification that replaces at least oneendogenous non-histidine codon of a human J_(H) segment with a histidinecodon, wherein the human J_(H) gene segment is selected from the groupconsisting of J_(H)1, J_(H)2, J_(H)3, J_(H)4, J_(H)5, J_(H)6, and acombination thereof.

In one embodiment, the added or substituted histidine codon is presentin a heavy chain variable region nucleotide sequence that encodes partof a CDR3. In one embodiment, the part of CDR3 comprises an amino acidsequence derived from a reading frame of a genetically modified human Dgene segment comprising a modification that replaces at least oneendogenous non-histidine codon in the reading frame with a histidinecodon.

In one embodiment, the endogenous non-histidine codon that issubstituted with a histidine codon encodes the amino acid selected fromY, N, D, Q, S, W, and R.

In one embodiment, the added or substituted histidine codon is presentin at least one reading frame of the human D gene segment that is mostfrequently observed in VELOCIMMUNE® humanized immunoglobulin mice.

In one embodiment, the reading frame of the genetically modified human Dgene segment that encodes part of CDR3 is selected from a hydrophobicframe, a stop frame, and a hydrophilic frame.

In one embodiment, the reading frame is a hydrophobic frame of a human Dgene segment.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D1-1 (GTTGT; SEQ ID NO: 88), D1-7(GITGT; SEQ ID NO: 89), D1-20 (GITGT; SEQ ID NO: 89), and D1-26 (GIVGAT;SEQ ID NO:90), and the human D gene segment further comprises amodification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D2-2 (DIVVVVPAAI; SEQ ID NO:92),D2-8 (DIVLMVYAI; SEQ ID NO: 94), D2-15 (DIVVVVAAT; SEQ ID NO:95), andD2-21 (HIVVVVTAI; SEQ ID NO: 97), and the human D gene segment furthercomprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D3-3 (ITIFGVVII; SEQ ID NO:98),D3-9 (ITIF*LVII; SEQ ID NO:99, SEQ ID NO:100), D3-10 (ITMVRGVII; SEQ IDNO:101), D3-16 (IMITFGGVIVI; SEQ ID NO:102), and D3-22 (ITMIVVVIT; SEQID NO:103), and the human D gene segment further comprises amodification that replaces at least one endogenous codon in thenucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D4-4 (TTVT; SEQ ID NO:105), D4-11(TTVT; SEQ ID NO:105), D4-17 (TTVT; SEQ ID NO:105), D4-23 (TTVVT; SEQ IDNO: 106) and the human D gene segment further comprises a modificationthat replaces at least one endogenous non-histidine codon in thenucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D5-5 (VDTAMV; SEQ ID NO: 107),D5-12 (VDIVATI; SEQ ID NO:108), D5-18 (VDTAMV; SEQ ID NO:107), and D5-24(VEMATI; SEQ ID NO:109), and the human D gene segment further comprisesa modification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the hydrophobic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D6-6 (SIAAR; SEQ ID NO:111), D6-13(GIAAAG; SEQ ID NO:113), and D6-19 (GIAVAG; SEQ ID NO:115), and thehuman D gene segment further comprises a modification that replaces atleast one endogenous non-histidine codon in the nucleotide sequence witha histidine codon.

In one embodiment, the hydrophobic frame comprises a nucleotide sequencethat encodes human D7-27 (LTG), and the human D gene segment furthercomprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the reading frame is a stop reading frame of a humanD gene segment.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D1-1 (VQLER; SEQ ID NO:8),D1-7(V*LEL), D1-20(V*LER), D1-26 (V*WELL; SEQ ID NO:12), and the human Dgene segment further comprises a modification that replaces at least oneendogenous non-histidine codon in the nucleotide sequence with ahistidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D2-2 (RIL**YQLLY; SEQ ID NO:14),D2-8 (RILY*WCMLY; SEQ ID NO:16 and SEQ ID NO: 17), D2-15 (RIL*WW*LLL),and D2-21 (SILWW*LLF; SEQ ID NO:19), and the human D gene segmentfurther comprises a modification that replaces at least one endogenousnon-histidine codon in the nucleotide sequence with a histidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D3-3 (VLRFLEWLLY; SEQ ID NO:21),D3-9 (VLRYFDWLL*; SEQ ID NO:23), D3-10 (VLLWFGELL*; SEQ ID NO:25), D3-16(VL*LRLGELSLY; SEQ ID NO:27), and D3-22 (VLL***WLLL; SEQ ID NO:29), andthe human D gene segment comprises a modification that replaces at leastone endogenous non-histidine codon in the nucleotide sequence with ahistidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D4-4 (*LQ*L), D4-11 (*LQ*L), D4-17(*LR*L), and D4-23 (*LRW*L), and the human D gene segment comprises amodification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D5-5 (WIQLWL; SEQ ID NO:35); D5-12(WI*WLRL; SEQ ID NO:37), D5-18 (WIQLWL; SEQ ID NO:35), and D5-24(*RWLQL; SEQ ID NO:39), and the human D gene segment comprises amodification that replaces at least one endogenous non-histidine codonin the nucleotide sequence with a histidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D6-6 (V*QLV), D6-13 (V*QQLV; SEQID NO:41), and D6-19 (V*QWLV; SEQ ID NO:43), and the human D genesegment further comprises a modification that replaces at least oneendogenous non-histidine codon in the nucleotide sequence with ahistidine codon.

In one embodiment, the stop reading frame of the human D gene segmentcomprises a nucleotide sequence that encodes D7-27 (*LG), and the humanD gene segment further comprises a modification that replaces at leastone endogenous codon of the human D gene segment in the nucleotidesequence with a histidine codon.

In one embodiment, the reading frame is a hydrophilic frame of a human Dgene segment.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D1-1 (YNWND; SEQ ID NO: 45), D1-7(YNWNY; SEQ ID NO: 47), D1-20 (YNWND; SEQ ID NO: 45), and D1-26 (YSGSYY;SEQ ID NO:49), and the human D gene segment further comprises amodification that replaces at least one endogenous codon in thenucleotide sequence with a histidine codon. In one embodiment, thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 46, SEQID NO: 48, SEQ ID NO: 50, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D2-2 (GYCSSTSCYT; SEQ ID NO:51),D2-8 (GYCTNGVCYT; SEQ ID NO: 53), D2-15 (GYCSGGSCYS; SEQ ID NO:55), andD2-21 (AYCGGDCYS; SEQ ID NO:57), and the human D gene segment furthercomprises a modification that replaces at least one endogenous codon inthe nucleotide sequence with a histidine codon. In one embodiment, thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 52, SEQID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D3-3 (YYDFWSGYYT; SEQ ID NO:59),D3-9 (YYDILTGYYN; SEQ ID NO:61), D3-10 (YYYGSGSYYN; SEQ ID NO:63), D3-16(YYDYVWGSYRYT; SEQ ID NO:65), and D3-22 (YYYDSSGYYY; SEQ ID NO:67), andthe human D gene segment further comprises a modification that replacesat least one endogenous codon in the nucleotide sequence with ahistidine codon. In one embodiment, the hydrophilic frame comprises anucleotide sequence encodes the amino acid sequence selected from thegroup consisting of SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ IDNO: 66, SEQ ID NO: 68, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D4-4 (DYSNY; SEQ ID NO:69), D4-11(DYSNY; SEQ ID NO:69), D4-17 (DYGDY; SEQ ID NO:71), and D4-23 (DYGGNS;SEQ ID NO:73), and the human D gene segment comprises a modificationthat replaces at least one endogenous codon in the nucleotide sequencewith a histidine codon. In one embodiment, the hydrophilic framecomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of SEQ ID NO: 70, SEQ ID NO: 72, SEQID NO: 74, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D5-5 (GYSYGY; SEQ ID NO:75), D5-12(GYSGYDY; SEQ ID NO:77), D5-18 (GYSYGY; SEQ ID NO:75), and D5-24(RDGYNY; SEQ ID NO:79), and the human D gene segment further comprises amodification that replaces at least one endogenous codon in thenucleotide sequence with a histidine codon. In one embodiment, thehydrophilic frame comprises a nucleotide sequence that encodes the aminoacid sequence selected from the group consisting of SEQ ID NO: 76, SEQID NO: 78, SEQ ID NO: 80, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of D6-6 (EYSSSS; SEQ ID NO: 81),D6-13 (GYSSSWY; SEQ ID NO:83), and D6-19 (GYSSGWY; SEQ ID NO:85), andthe human D gene segment further comprises a modification that replacesat least one endogenous codon in the nucleotide sequence with ahistidine codon. In one embodiment, the hydrophilic frame comprises anucleotide sequence that encodes the amino acid sequence selected fromthe group consisting of SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQID NO: 76, and a combination thereof.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes D7-27 (NWG), and the humanD gene segment further comprises a modification that replaces at leastone endogenous codon in the nucleotide sequence a histidine codon.

In one embodiment, the hydrophilic frame of the human D gene segmentcomprises a nucleotide sequence that encodes the amino acid sequenceselected from the group consisting of SEQ ID NO: 46, SEQ ID NO: 48, SEQID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58,SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO:68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ IDNO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, anda combination thereof.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprising the inverted human D gene segment isoperably linked to a human or non-human heavy chain constant regionnucleotide sequence that encodes an immunoglobulin isotype selected fromIgM, IgD, IgG, IgE, and IgA.

In one embodiment, the human unrearranged immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human ornon-human heavy chain constant region nucleotide sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In oneembodiment, the heavy chain constant region nucleotide sequencecomprises a C_(H)1, a hinge, a C_(H)2, and a C_(H)3(C_(H)1-hinge-C_(H)2-C_(H)3).

In one embodiment, a heavy chain constant region nucleotide sequence ispresent at an endogenous locus (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the heavy chain constant region nucleotide sequencecomprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a modification at position 250 (e.g., E or Q); 250 and 428(e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256(e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433(e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591(e.g., V259I), and a 308F (e.g., V308F) modification; a 433K (e.g.,H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g.,252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g.,T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or308P), wherein the modification increases the affinity of the heavychain constant region amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 252 and 257,wherein the modification increases the affinity of the human C_(H)2amino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 307 and 311,wherein the modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)3 amino acid sequence, wherein the C_(H)3 aminoacid sequence comprises at least one modification between amino acidresidues at positions 433 and 436, wherein the modification increasesthe affinity of the C_(H)3 amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,N434S, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,V259I, V308F, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising an N434A mutation.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M252Y,S254T, T256E, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of T250Q,M248L, or both.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of H433K,N434Y, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises: (1) a first allele, wherein the unrearranged humanimmunoglobulin heavy chain variable region nucleotide sequence asdescribed herein is operably linked to a first heavy chain constantregion nucleotide sequence encoding a first CH₃ amino acid sequence of ahuman IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and(2) a second allele, wherein the unrearranged human immunoglobulin heavychain variable region nucleotide sequence as described herein isoperably linked to a second heavy chain constant region nucleotidesequence encoding a second C_(H)3 amino acid sequence of the human IgGselected from IgG1, IgG2, IgG4, and a combination thereof, and whereinthe second CH₃ amino acid sequence comprises a modification that reducesor eliminates binding for the second CH₃ amino acid sequence to ProteinA (see, for example, US 2010/0331527A1, incorporated by reference hereinin its entirety).

In one embodiment, the second CH₃ amino acid sequence comprises an H95Rmodification (by IMGT exon numbering; H435R by EU numbering). In oneembodiment the second C_(H)3 amino acid sequence further comprises anY96F modification (by IMGT exon numbering; H436F by EU). In anotherembodiment, the second CH₃ amino acid sequence comprises both an H95Rmodification (by IMGT exon numbering; H435R by EU numbering) and an Y96Fmodification (by IMGT exon numbering; H436F by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG1 and further comprises a mutation selected from the groupconsisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L38M,N384S, K392N, V397M, and V422I by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG2 and further comprises a mutation selected from the groupconsisting of N44S, K52N, and V82I (IMGT: N384S, K392N, and V422I byEU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG4 and further comprises a mutation selected from the groupconsisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT: Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

In one embodiment, the heavy chain constant region amino acid sequenceis a non-human constant region amino acid sequence, and the heavy chainconstant region amino acid sequence comprises one or more of any of thetypes of modifications described above.

In one embodiment, the heavy chain constant region nucleotide sequenceis a human heavy chain constant region amino acid sequence, and thehuman heavy chain constant region amino acid sequence comprises one ormore of any of the types of modifications described above.

In one embodiment, all or substantially all endogenous V_(H), D, andJ_(H) gene segments are deleted from an immunoglobulin heavy chain locusor rendered non-functional (e.g., via insertion of a nucleotide sequence(e.g., an exogenous nucleotide sequence) in the immunoglobulin locus orvia non-functional rearrangement, or inversion, of the endogenous V_(H),D, J_(H) segments). In one embodiment, e.g., about 80% or more, about85% or more, about 90% or more, about 95% or more, about 96% or more,about 97% or more, about 98% or more, or about 99% or more of allendogenous V_(H), D, or J_(H) gene segments are deleted or renderednon-functional. In one embodiment, e.g., at least 95%, 96%, 97%, 98%, or99% of endogenous functional V, D, or J gene segments are deleted orrendered non-functional.

In one embodiment, the genetically modified locus comprises amodification that deletes or renders non-functional all or substantiallyall endogenous V_(H), D, and J_(H) gene segments; and the genomic locuscomprises the genetically modified, unrearranged human heavy chainvariable region nucleotide sequence comprising a substitution of atleast one endogenous non-histidine codon with a histidine codon in atleast one reading frame. In one embodiment, the genetically modified,unrearranged immunoglobulin heavy chain variable gene sequence ispresent at an endogenous location (i.e., where the nucleotide sequenceis located in a wild-type non-human animal) or present ectopically(e.g., at a locus different from the endogenous immunoglobulin chainlocus in its genome or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the genetically modified locus comprises anendogenous Adam6a gene, Adam6b gene, or both, and the geneticmodification does not affect the expression and/or function of theendogenous Adam6a gene, Adam6b gene, or both.

In one embodiment, the genetically modified locus comprises anectopically present Adam6a gene, Adam6b gene, or both. In oneembodiment, the Adam6a gene is a non-human Adam6a gene. In oneembodiment, the Adam6a gene is a mouse Adam6a gene. In one embodiment,the Adam6a gene is a human Adam6a gene. In one embodiment, the Adam6bgene is a non-human Adam6b gene. In one embodiment, the Adam6b gene is amouse Adam6b gene. In one embodiment, the Adam6b gene is a human Adam6bgene.

In one embodiment, the genetically modified immunoglobulin locus furthercomprises a humanized, unrearranged λ and/or κ light chain variable genesequence. In one embodiment, the humanized, unrearranged λ and/or κlight chain variable gene sequence is operably linked to animmunoglobulin light chain constant region nucleotide sequence selectedfrom a λ light chain constant region nucleotide sequence and a κ lightchain constant region nucleotide sequence. In one embodiment, thehumanized, unrearranged λ light chain variable region nucleotidesequence is operably linked to a λ light chain constant regionnucleotide sequence. In one embodiment, the λ light chain constantregion nucleotide sequence is a mouse, rat, or human sequence. In oneembodiment, the humanized, unrearranged κ light chain variable regionnucleotide sequence is operably linked to a κ light chain constantregion nucleotide sequence. In one embodiment, the κ light chainconstant region nucleotide sequence is a mouse, rat, or human sequence.

In one embodiment, the genetically modified immunoglobulin locuscomprises an unrearranged light chain variable gene sequence thatcontains at least one modification that introduces at least onehistidine codon in at least one reading frame encoding a light chainvariable domain. In one embodiment, the genetically modifiedimmunoglobulin locus comprises a rearranged (e.g., a rearranged λ or κV/J sequence) sequence that comprises one, two, three, or four codonsfor histidine in a light chain CDR. In one embodiment, the CDR is aselected from a CDR1, CDR2, CDR3, and a combination thereof. In oneembodiment, the unrearranged or rearranged light chain variable regionnucleotide sequence is an unrearranged or rearranged human λ or κ lightchain variable region nucleotide sequence. In one embodiment, theunrearranged or rearranged human λ or κ light chain variable regionnucleotide sequence is present at an endogenous mouse immunoglobulinlight chain locus. In one embodiment, the mouse immunoglobulin lightchain locus is a mouse κ locus. In one embodiment the mouseimmunoglobulin light chain locus is a mouse λ locus.

In one embodiment, the genetically modified immunoglobulin locus asdescribed herein is present in an immunoglobulin heavy chain locus of amouse. In one embodiment, the genetically modified immunoglobulin locusis present in a humanized immunoglobulin heavy chain locus in aVELOCIMMUNE® mouse.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein exhibits a weaker antigen bindingat an acidic environment (e.g., at a pH of about 5.5 to about 6.0) thana corresponding wild-type heavy chain variable domain without thegenetic modification.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification.

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises a B cell population that, upon stimulationwith an antigen of interest, is capable of producing antigen-bindingproteins, e.g., antibodies, comprising a heavy chain variable domaincomprising one or more histidine residues. The antigen-binding proteinsas described herein when administered into a subject, exhibits anincreased serum half-life over a corresponding wild-type antigen-bindingprotein, which possesses a similar or sufficiently similar amino acidsequence that encodes the heavy chain variable domain but does notcomprise a histidine residue in the heavy chain variable domain. In someembodiments, the antigen-binding protein described herein exhibits anincreased serum half-life that is at least about 2-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold higher than the corresponding wild-type antigen-binding protein,which possesses a similar or sufficiently similar amino acid sequencethat encodes the heavy chain variable domain but does not comprise ahistidine residue in the heavy chain variable domain.

In one aspect, a method for making a non-human animal comprising agenetically modified immunoglobulin heavy chain variable locus isprovided, comprising: (a) modifying a genome of a non-human animal todelete or render non-functional endogenous immunoglobulin heavy chain V,D, and J gene segments (e.g., via insertion of a nucleotide sequence(e.g., an exogenous nucleotide sequence) in the immunoglobulin locus orvia non-functional rearrangement or inversion of endogenous V_(H), D,J_(H) segments); and (b) placing in the genome a human V_(H), D, andJ_(H) gene segment, wherein at least one of the human D gene segment hasbeen inverted 5′ to 3′ with respect to a corresponding wild-typesequence, and wherein at least one reading frame of the inverted human Dgene segment comprises a histidine codon.

In one embodiment, the non-human animal is a mammal, including a rodent,e.g., a mouse, a rat, or a hamster

In one embodiment, the genetically modified immunoglobulin locus ispresent in a germline genome.

In one embodiment, the genetically modified immunoglobulin locus encodesan immunoglobulin heavy chain variable domain comprising one or more, 2or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 ormore, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 ormore, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 ormore, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 ormore, 27 or more, 28 or more, 29 or more, 30 or more, 31 or more, 32 ormore, 33 or more, or 34 or more of histidine residues.

In one embodiment, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least nine, atleast ten, at least eleven, at least twelve, at least thirteen, at leastfourteen, at least fifteen, at least sixteen, at least seventeen, atleast eighteen, at least nineteen, at least twenty, at least twenty one,at least twenty two, at least twenty three, at least twenty four, or allor substantially all of functional human D gene segments have invertedorientation with respect to corresponding wild type sequences.

In one embodiment, all or substantially all of endogenous immunoglobulinV_(H), D, J_(H) gene segments are deleted from the immunoglobulin heavychain locus or rendered non-functional (e.g., via insertion of anucleotide sequence, e.g., exogenous nucleotide sequence, in theimmunoglobulin locus or via non-functional rearrangement or inversion ofall, or substantially all, endogenous immunoglobulin V_(H), D, J_(H)segments), and the genetically modified immunoglobulin locus comprises ahuman V_(H), D, and J_(H) gene segments, wherein at least one of thehuman D gene segment is present in an inverted orientation with respectto a corresponding wild type sequence, and wherein at least one readingframe in the inverted human D gene segment comprises at least onehistidine codon.

In one embodiment, the inverted human D gene segment is operably linkedto a human V_(H) gene segment, and/or human J_(H) gene segment

In one embodiment, the human D gene segment that is present in theinverted orientation relative to wild type sequences is selected fromthe group consisting of D1-1, D1-7, D1-20, D1-26, D2-2, D2-8, D2-15,D2-21, D3-3, D3-9, D3-10, D3-16, D3-22, D4-4, D4-11, D4-17, D4-23, D5-5,D5-12, D5-18, D5-24, D6-6, D6-13, D6-19, D7-27, and a combinationthereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD1 gene segment selected from the group consisting of D1-1, D1-7, D1-20,D1-26, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD2 gene segment selected from the group consisting of D2-2, D2-8, D2-15,D2-21, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD3 gene segment selected from the group consisting of D3-3, D3-9, D3-10,D3-16, D3-22, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD4 gene segment selected from the group consisting of D4-4, D4-11,D4-17, D4-23, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD5 gene segment selected from the group consisting of D5-5, D5-12,D5-18, D5-24, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence is aD6 gene segment selected from the group consisting of D6-6, D6-13,D6-19, and a combination thereof.

In one embodiment, the human D gene segment that is present in theinverted orientation relative to a corresponding wild type sequence isD7-27.

In one embodiment, the reading frame of the human D gene segment isselected from a stop reading frame, a hydrophilic reading frame, ahydrophobic reading frame, and a combination thereof.

In one embodiment, the unrearranged heavy chain variable regionnucleotide sequence comprising the inverted human D gene segment isoperably linked to a human or non-human heavy chain constant regionnucleotide sequence that encodes an immunoglobulin isotype selected fromIgM, IgD, IgG, IgE, and IgA.

In one embodiment, the human unrearranged immunoglobulin heavy chainvariable region nucleotide sequence is operably linked to a human ornon-human heavy chain constant region nucleotide sequence selected froma C_(H)1, a hinge, a C_(H)2, a C_(H)3, and a combination thereof. In oneembodiment, the heavy chain constant region nucleotide sequencecomprises a C_(H)1, a hinge, a C_(H)2, and a C_(H)3(C_(H)1-hinge-C_(H)2-C_(H)3).

In one embodiment, a heavy chain constant region nucleotide sequence ispresent at an endogenous locus (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the heavy chain constant region nucleotide sequencecomprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a modification at position 250 (e.g., E or Q); 250 and 428(e.g., L or F); 252 (e.g., L/Y/F/W or T), 254 (e.g., S or T), and 256(e.g., S/R/Q/E/D or T); or a modification at position 428 and/or 433(e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or a modification atposition 250 and/or 428; or a modification at position 307 or 308 (e.g.,308F, V308F), and 434. In one embodiment, the modification comprises a428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591(e.g., V259I), and a 308F (e.g., V308F) modification; a 433K (e.g.,H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g.,252Y, 254T, and 256E) modification; a 250Q and 428L modification (e.g.,T250Q and M428L); and a 307 and/or 308 modification (e.g., 308F or308P), wherein the modification increases the affinity of the heavychain constant region amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 252 and 257,wherein the modification increases the affinity of the human C_(H)2amino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 307 and 311,wherein the modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)3 amino acid sequence, wherein the C_(H)3 aminoacid sequence comprises at least one modification between amino acidresidues at positions 433 and 436, wherein the modification increasesthe affinity of the C_(H)3 amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,N434S, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M428L,V259I, V308F, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising an N434A mutation.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of M252Y,S254T, T256E, and a combination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of T250Q,M248L, or both.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human heavy chain constant region amino acid sequencecomprising a mutation selected from the group consisting of H433K,N434Y, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises: (1) a first allele, wherein the unrearranged humanimmunoglobulin heavy chain variable region nucleotide sequence asdescribed herein is operably linked to a first heavy chain constantregion nucleotide sequence encoding a first CH₃ amino acid sequence of ahuman IgG selected from IgG1, IgG2, IgG4, and a combination thereof; and(2) a second allele, wherein the unrearranged human immunoglobulin heavychain variable region nucleotide sequence as described herein isoperably linked to a second heavy chain constant region nucleotidesequence encoding a second C_(H)3 amino acid sequence of the human IgGselected from IgG1, IgG2, IgG4, and a combination thereof, and whereinthe second CH₃ amino acid sequence comprises a modification that reducesor eliminates binding for the second CH₃ amino acid sequence to ProteinA (see, for example, US 2010/0331527A1, incorporated by reference hereinin its entirety).

In one embodiment, the second CH₃ amino acid sequence comprises an H95Rmodification (by IMGT exon numbering; H435R by EU numbering). In oneembodiment the second CH₃ amino acid sequence further comprises an Y96Fmodification (by IMGT exon numbering; H436F by EU). In anotherembodiment, the second CH₃ amino acid sequence comprises both an H95Rmodification (by IMGT exon numbering; H435R by EU numbering) and an Y96Fmodification (by IMGT exon numbering; H436F by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG1 and further comprises a mutation selected from the groupconsisting of D16E, L18M, N44S, K52N, V57M, and V82I (IMGT; D356E, L38M,N384S, K392N, V397M, and V422I by EU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG2 and further comprises a mutation selected from the groupconsisting of N44S, K52N, and V82I (IMGT: N384S, K392N, and V422I byEU).

In one embodiment, the second CH₃ amino acid sequence is from a modifiedhuman IgG4 and further comprises a mutation selected from the groupconsisting of Q15R, N44S, K52N, V57M, R69K, E79Q, and V82I (IMGT: Q355R,N384S, K392N, V397M, R409K, E419Q, and V422I by EU).

In one embodiment, the heavy chain constant region amino acid sequenceis a non-human constant region amino acid sequence, and the heavy chainconstant region amino acid sequence comprises one or more of any of thetypes of modifications described above.

In one embodiment, the heavy chain constant region nucleotide sequenceis a human heavy chain constant region amino acid sequence, and thehuman heavy chain constant region amino acid sequence comprises one ormore of any of the types of modifications described above.

In one embodiment, all or substantially all endogenous V_(H), D, andJ_(H) gene segments are deleted from an immunoglobulin heavy chain locusor rendered non-functional (e.g., via insertion of a nucleotide sequence(e.g., an exogenous nucleotide sequence) in the immunoglobulin locus orvia non-functional rearrangement, or inversion, of the endogenous V_(H),D, J_(H) segments). In one embodiment, e.g., about 80% or more, about85% or more, about 90% or more, about 95% or more, about 96% or more,about 97% or more, about 98% or more, or about 99% or more of allendogenous V_(H), D, or J_(H) gene segments are deleted or renderednon-functional. In one embodiment, e.g., at least 95%, 96%, 97%, 98%, or99% of endogenous functional V, D, or J gene segments are deleted orrendered non-functional.

In one embodiment, the genetically modified immunoglobulin heavy chainlocus comprises a modification that deletes or renders, all orsubstantially all, non-functional endogenous V_(H), D, and J_(H) genesegments; and the genetically modified locus comprises an unrearrangedheavy chain variable region nucleotide sequence comprising at least oneinverted human D gene segment as described herein wherein theunrearranged heavy chain variable region nucleotide sequence is presentat an endogenous location (i.e., where the nucleotide sequence islocated in a wild-type non-human animal) or present ectopically (e.g.,at a locus different from the endogenous immunoglobulin chain locus inits genome, or within its endogenous locus, e.g., within animmunoglobulin variable locus, wherein the endogenous locus is placed ormoved to a different location in the genome).

In one embodiment, the genetically modified immunoglobulin locuscomprises an endogenous Adam6a gene, Adam6b gene, or both, and thegenetic modification does not affect the expression and/or function ofthe endogenous Adam6a gene, Adam6b gene, or both.

In one embodiment, the genetically modified immunoglobulin locuscomprises an ectopically present Adam6a gene, Adam6b gene, or both. Inone embodiment, the Adam6a gene is a non-human Adam6a gene. In oneembodiment, the Adam6a gene is a mouse Adam6a gene. In one embodiment,the Adam6a gene is a human Adam6a gene. In one embodiment, the Adam6bgene is a non-human Adam6b gene. In one embodiment, the Adam6b gene is amouse Adam6b gene. In one embodiment, the Adam6b gene is a human Adam6bgene.

In one embodiment, the genetically modified immunoglobulin locus furthercomprises a humanized, unrearranged λ and/or κ light chain variable genesequence. In one embodiment, the humanized, unrearranged λ and/or κlight chain variable gene sequence is operably linked to animmunoglobulin light chain constant region nucleotide sequence selectedfrom a λ light chain constant region nucleotide sequence and a κ lightchain constant region nucleotide sequence. In one embodiment, thehumanized, unrearranged λ light chain variable region nucleotidesequence is operably linked to a λ light chain constant regionnucleotide sequence. In one embodiment, the λ light chain constantregion nucleotide sequence is a mouse, rat, or human sequence. In oneembodiment, the humanized, unrearranged κ light chain variable regionnucleotide sequence is operably linked to a κ light chain constantregion nucleotide sequence. In one embodiment, the κ light chainconstant region nucleotide sequence is a mouse, rat, or human sequence.

In one embodiment, the genetically modified immunoglobulin locuscomprises an unrearranged light chain variable gene sequence thatcontains at least one modification that introduces at least onehistidine codon in at least one reading frame encoding a light chainvariable domain. In one embodiment, the genetically modifiedimmunoglobulin locus comprises a rearranged (e.g., a rearranged λ or κV/J sequence) sequence that comprises one, two, three, or four codonsfor histidine in a light chain CDR. In one embodiment, the CDR is aselected from a CDR1, CDR2, CDR3, and a combination thereof. In oneembodiment, the unrearranged or rearranged light chain variable regionnucleotide sequence is an unrearranged or rearranged human λ or κ lightchain variable region nucleotide sequence. In one embodiment, theunrearranged or rearranged human λ or κ light chain variable regionnucleotide sequence is present at an endogenous mouse immunoglobulinlight chain locus. In one embodiment, the mouse immunoglobulin lightchain locus is a mouse κ locus. In one embodiment, the mouseimmunoglobulin light chain locus is a mouse immunoglobulin light chainlocus is a mouse λ locus.

In one embodiment, the genetically modified immunoglobulin locus asdescribed herein is present in an immunoglobulin heavy chain locus of amouse. In one embodiment, the genetically modified immunoglobulin locusis present in a humanized immunoglobulin heavy chain locus in aVELOCIMMUNE® mouse.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein exhibits a weaker antigen bindingat an acidic environment (e.g., at a pH of about 5.5 to about 6.0) thana corresponding wild-type heavy chain variable domain without thegenetic modification.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification.

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises an enriched B cell population that, uponstimulation with an antigen of interest, is capable of producingantigen-binding proteins, e.g., antibodies, comprising a heavy chainvariable domain comprising one or more histidine residues. Theantigen-binding proteins as described herein when administered into asubject, exhibits an increased serum half-life over a correspondingwild-type antigen-binding protein, which possesses a similar orsufficiently similar amino acid sequence that encodes the heavy chainvariable domain but does not comprise a histidine residue in the heavychain variable domain. In some embodiments, the antigen-binding proteindescribed herein exhibits an increased serum half-life that is at leastabout 2-fold, at least about 5-fold, at least about 10-fold, at leastabout 15-fold, at least about 20-fold higher than the correspondingwild-type antigen-binding protein, which possesses a similar orsufficiently similar amino acid sequence that encodes the heavy chainvariable domain but does not comprise a histidine residue in the heavychain variable domain.

In one aspect, a method for making a non-human animal that is capable ofproducing an immunoglobulin heavy chain variable domain with enhancedserum half-life and/or enhanced pH-dependent recyclability is provided,comprising (a) modifying a genome of a non-human animal to delete orrender non-functional endogenous immunoglobulin heavy chain V, D, and Jgene segments (e.g., via insertion of a nucleotide sequence (e.g., anexogenous nucleotide sequence) in the immunoglobulin locus or vianon-functional rearrangement or inversion of endogenous V_(H), D, J_(H)segments); and (b) placing in the genome an unrearranged human heavychain variable region nucleotide sequence, wherein the unrearrangedheavy chain variable region nucleotide sequence comprises an addition ofleast one histidine codon or a substitution of at least one endogenousnon-histidine codon with a histidine codon, and wherein anantigen-binding protein comprising the immunoglobulin heavy chainvariable domain produced by the non-human animal exhibits enhanced serumhalf-life and/or enhanced pH-dependent recyclability as compared to awild-type immunoglobulin heavy chain domain.

In one embodiment, the non-human animal, upon contact with an antigen,can produce an enriched population of B cell repertoire that expressesan antigen-binding protein with enhanced serum half-life and/or enhancedpH-dependent recyclability, wherein the enriched B cell populationcomprises any genetic modifications as described herein.

In one embodiment, an antigen-binding protein produced by thegenetically modified non-human animal is characterized by sufficientaffinity to an antigen of interest at a neutral pH (e.g., pH of about7.0 to about 7.4) and enhanced dissociation of the antibody from anantigen-antigen-binding protein complex at a pH less than the neutral pH(e.g., at an endosomal pH, e.g. pH of about 5.5 to 6.0).

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinlocus as described herein is characterized by improved pH-dependentrecyclability, enhanced serum half-life, or both as compared with awild-type antigen-binding protein without the genetic modification.

In one embodiment, the genetically modified immunoglobulin locusdescribed herein comprises a an enriched B cell population that, uponstimulation with an antigen of interest, is capable of producingantigen-binding proteins, e.g., antibodies, comprising a heavy chainvariable domain comprising one or more histidine residues. Theantigen-binding proteins as described herein when administered into asubject, exhibits an increased serum half-life over a correspondingwild-type antigen-binding protein, which possesses a similar orsufficiently similar amino acid sequence that encodes the heavy chainvariable domain but does not comprise a histidine residue in the heavychain variable domain. In some embodiments, the antigen-binding proteindescribed herein exhibits an increased serum half-life that is at leastabout 2-fold, at least about 5-fold, at least about 10-fold, at leastabout 15-fold, at least about 20-fold higher than the correspondingwild-type antigen-binding protein, which possesses a similar orsufficiently similar amino acid sequence that encodes the heavy chainvariable domain but does not comprise a histidine residue in the heavychain variable domain.

In one embodiment, the antigen-binding protein comprises animmunoglobulin heavy chain variable domain that is capable ofspecifically binding an antigen of interest with an affinity (K_(D))lower than 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, and 10⁻¹² at a neutralpH (pH of about 7.0 to about 7.4).

In one aspect, a method for obtaining an antigen-binding protein withenhanced recyclability and/or improved serum half-life is provided,comprising: (a) immunizing a non-human animal having a geneticallymodified immunoglobulin locus as described herein wherein the non-humananimal comprises an unrearranged human heavy chain variable regionnucleotide sequence comprising an addition of least one histidine codonor a substitution of at least one endogenous non-histidine codon with ahistidine codon; (b) allowing the non-human animal to mount an immuneresponse; (c) harvesting a lymphocyte (e.g., a B cell) from theimmunized non-human animal; (d) fusing the lymphocyte with a myelomacell to form a hybridoma cell, and (e) obtaining an antigen-bindingprotein produced by the hybridoma cell, wherein the antigen-bindingprotein exhibits enhanced recyclability and/or serum stability.

In one aspect, a genetically modified immunoglobulin heavy chain locusobtainable by any of the methods as described herein is provided.

In one aspect, a genetically modified non-human animal obtainable by anyof the methods as described herein is provided.

In various embodiments, the non-human animal is a mammal. In oneembodiment, the mammal is a rodent, e.g., a mouse, a rat, or a hamster.

In various embodiments, the genetically modified immunoglobulin loci asdescribed herein are present in the germline genome of a non-humananimal, e.g., a mammal, e.g., a rodent, e.g., a mouse, a rat, or ahamster.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B illustrate the amino acid sequences encoded by the threereading frames (i.e., stop, hydrophilic, and hydrophobic reading frames)of human D gene segments (D) and the amino acid sequences encoded by thethree reading frames of histidine-substituted human D gene segments(HD). Introduction of histidine codons (typed in bold) in thehydrophilic reading frame also changed many stop codons in the stopreading frame to Ser codons (typed in bold) but introduced few changesin the hydrophobic reading frame. The “*” symbol represents a stopcodon, and the comma between the two SEQ ID NOs indicates that there aretwo amino acid sequences separated by the stop codon.

FIG. 2 illustrates schemes for targeting pLMa0174 containing aspectinomycin selection cassette into the 5′ end of MAID 1116 (Step 1.BHR (Spec)). In Step 1, a chloramphenicol selection cassette, a neomycinselection cassette, a loxP site, two V_(H) gene segments (hV_(H)1-3 andhV_(H)1-2), the human Adam6 gene, all of which are located upstream ofhV_(H)6-1, were deleted from the clone and replaced by a spectinomycincassette to yield the VI433 clone. In Step 2 (BHR (Hyg+Spec)), pNTu0002containing a hygromycin cassette flanked by FRT sites was targeted intoa region comprising human immunoglobulin D gene segments. Via Step 2,all human D gene segments were deleted from VI433 and replaced with thehygromycin cassette to yield MAID6011 VI 434 (clone 1).

FIG. 3 illustrates schemes for assembling histidine-substituted human Dgene segments via sequential ligation.

FIG. 4 illustrates the introduction of pre-assembled,histidine-substituted human D gene segments containing a neomycincassette into a region between the most D-proximal V_(H) gene segment(V_(H)6-1) and the most D-proximal J_(H) gene segment (J_(H)1) viaenzyme-mediated digestion (PI-Scel and I-Ceul) and ligation. Thisprocess removes the hygromycin cassette from MAID 6011 VI434 andintroduces pre-assembled human histidine-substituted D gene segmentsinto the clone. Bacterial cells comprising a successfully targeted cloneare selected based on both neomycin and spectinomycin resistance. Theresulting clone (MAID6012 VI469) comprises, from 5′ to 3′, (1) aspectinomycin selection cassette, (2) a 50 kb arm comprising a humanV_(H) gene segment (V_(H)6-1), (3) a neomycin cassette flanked by loxPsites, (4) human D gene segments containing histidine substitutions (HD1.1-6.6 (9586 bp; SEQ ID NO: 1), HD 1.7-6.13 (9268 bp; SEQ ID NO: 2), HD1.14-6.19 (9441 bp; SEQ ID NO: 3), and HD 1.20-6.25, 1.26 (11592 bp; SEQID NO: 4)), (5) about 25 kb of a genomic region containing human J_(H)gene segments, (6) a mouse E_(i) sequence (SEQ ID NO: 5; an intronicenhancer that promotes V_(H) to DJ_(H) rearrangement in developing Bcells), and (7) a mouse IgM constant region nucleotide sequence (mIgMexon 1; SEQ ID NO: 7).

FIG. 5 illustrates schemes for deleting the human immunoglobulin heavychain D gene region from the MAID 1460 heterozygous ES cells bytargeting the 129 strain-derived chromosome of MAID 1460 het with thehygromycin selection cassette in MAID 6011 VI434.

FIG. 6 shows a list of primers and probes used to confirm a loss ofallele (LOA), a gain of allele (GOA), or a parental allele (Parental) inthe screening assays for identifying MAID 6011

FIG. 7 illustrates schemes for constructing MAID 6012 het by targetingMAID 6011 heterozygous ES cells with MAID 6012 VI469. Electroporation ofthe MAID 6012 VI469 construct into the MAID 6011 heterozygous ES cellsyielded MAID 6012 heterozygous ES cells in which the 129 strain-derivedchromosome is modified to contain, from 5′ to 3′ direction, an FRT site,human V_(H) gene segments, a mouse genomic region comprising adam6genes, a floxed neomycin selection cassette, human D gene segmentscomprising histidine substitutions (HD 1.1-6.6 (9586 bp; SEQ ID NO: 1),HD 1.7-6.13 (9268 bp; SEQ ID NO: 2), HD 1.14-6.19 (9441 bp; SEQ ID NO:3), and HD 1.20-6.25, 1.26 (11592 bp; SEQ ID NO: 4)), human J_(H) genesegments, a mouse E_(i) sequence (SEQ ID NO: 5; an intronic enhancerthat promotes V_(H) to DJ_(H) rearrangement in developing B cells), anda mouse IgM constant region nucleotide sequence (mIgM exon 1; SEQ ID NO:7).

FIG. 8 shows a list of primers and probes used to confirm a loss ofallele (LOA), a gain of allele (GOA), or a parental allele (Parental) inthe screening assay for identifying MAID 6012.

FIG. 9 illustrates schemes for removing a neomycin cassette from MAID6012 heterozygous ES cells. Electroporation of a Cre-expressing plasmidinto the MAID 6012 ES cells lead to recombination and deletion of thefloxed neomycin cassette, yielding MAID 6013 heterozygous ES cells.

FIGS. 10A-10E illustrate human D gene segment nucleotide sequences withtranslations for each of the six reading frames, i.e., three readingframes for direct 5′ to 3′ orientation and three reading frames forinverted orientation (3′ to 5′ orientation). The “*” symbol represents astop codon, and the comma between two SEQ ID NOs indicates that thereare two amino acid sequences separated by the stop codon.

FIGS. 11-13 illustrate mRNA sequences and their encoded proteinsequences expressed by 6013 F0 heterozygous mice, which comprisehistidine-substituted human D gene segments (HD 1.1-6.6 (9586 bp; SEQ IDNO: 1), HD 1.7-6.13 (9268 bp; SEQ ID NO: 2), HD 1.14-6.19 (9441 bp; SEQID NO: 3), and HD 1.20-6.25, 1.26 (11592 bp; SEQ ID NO: 4)) in theimmunoglobulin heavy chain locus in their 129 strain-derived chromosome.The boxed sequences in each figure indicate the presence of histidinecodons in the CDR3 sequences derived from the genetically modifiedimmunoglobulin heavy chain locus comprising the histidine-substitutedhuman D gene segments. FWR represents frame region and CDR representscomplementarity determining region. In the alignment, the dot “.”indicates a sequence identical to the query sequence, and the dash “-”indicates a gap in the sequence.

FIG. 14 illustrates histidine incorporation frequency in immunoglobulinheavy chain CDR3 sequences. The X-axis represents the number ofhistidine codons appeared in each CDR3 sequence, and the Y-axisrepresents the corresponding proportion of reads. The “6013 F0 het”indicates CDR3 sequences expressed by the 6013 heterozygous micecomprising histidine-substituted D gene segments. The “VI3-Adam6”indicates CDR3 sequences obtained from control mice comprising human VH,D, and JH gene segments without the histidine modification as describedherein. The “ASAP” indicates CDR3 sequences obtained from the Regeneronantibody database, which was used as another control.

FIG. 15 illustrates an amino acid alignment of human Vκ1-39-derivedlight chains from various antigen-specific antibodies (A-K antibodies).Histidine (H) residues located within each light chain sequence are inbold. Various light chain regions (Framework and CDR) are indicatedabove the alignment.

FIG. 16 illustrates the combinations and locations of histidine residuesengineered in the CDR3 region of human Vκ1-39-derived light chains bymutagenesis. Corresponding nucleic acid sequences are included.Histidine residues introduced through mutagenesis and correspondingnucleic acid residues are shown in bold. Amino acid positions (105, 106,etc.) are based on a unique numbering described in Lefranc et al. (2003)Dev. Comp. Immunol. 27:55-77, and can also be viewed on www.imgt.org.

FIG. 17 illustrates the level of antibody expression in ng/mL detectedin the supernatants of CHO cells transfected with nucleic acids encodingfive (1-5) different heavy chains and Vκ1-39-derived light chains havinghistidine residues engineered at indicated locations (see Y axis) in theCDR3.

FIG. 18 is a western blot showing expression of selectedantigen-specific human antibodies containing histidine engineered lightchains in CHO cell supernatants.

FIGS. 19A-19J shows the binding kinetics for selected heavy chains fromantigen-specific antibodies paired with various histidine engineeredlight chains at a neutral (7.4) and acidic (5.5) pH.

FIGS. 20A-20E show the binding kinetics for selected heavy chains (1-5)from antigen-specific antibodies paired with various histidineengineered light chains at a neutral (7.4) and acidic (5.75) pH. Variouskinetic parameters including k_(a), k_(d), K_(D), and t_(1/2) are shown.NB=no binding.

FIG. 21 shows kinetic parameters (K_(D) and t_(1/2)) for antibodiescomprising parental universal light chain or histidine-modifieduniversal light chain paired with indicated heavy chains (2, 3, and 6).Histidine substitutions lead to strong pH dependence in severalantibodies. Histidine substitutions were made in CDR3 to convert thesequence ₁₀₅QQSYSTP₁₁₁ (SEQ ID NO:3) to ₁₀₅HHSYSTH₁₁₁ (SEQ ID NO:329).Note that NB=no binding detected (K_(D)>10 micromolar).

FIG. 22 shows the sequence and properties (% GC content, N, % mismatch,Tm) of selected mutagenesis primers used to engineer histidine residuesinto CDR3 of a rearranged human Vκ1-39/Jκ5 light chain sequence. SEQ IDNOs for these primers used in the Sequence Listing are included in theTable below. F=forward primer, R=reverse primer.

FIGS. 23A-23B show a general strategy for construction of targetingvectors for engineering of histidine residues into a rearranged humanlight chain variable region sequence derived from Vκ1-39/Jκ5 variableregion for making a genetically modified mouse that expresses antibodiescontaining the modified human light chain. FIGS. 23C-23D showintroduction of the targeting vector for ULC-H105/106/108/111substitutions into ES cells and generation of heterozygous mice from thesame; while FIGS. 23E-23F show introduction of the targeting vector forULC-H106/108/111 substitutions into ES cells and generation ofheterozygous mice from the same. The diagrams are not presented toscale. Unless indicated otherwise, filled shapes and solid linesrepresent mouse sequence, empty shapes and double lines represent humansequence.

FIG. 24 shows antiserum titers against immunogen from mice heterozygousfor histidine universal light chain (HULC) (with 4 Hissubstitutions—HULC 1927 mice; with 3 His substitutions—HULC 1930 mice)and wild type animals in a second bleed.

FIG. 25 is a comparison of the number of total antigen positive clonesand the number of antigen positive clones displaying pH sensitiveantigen binding obtained from hybridoma fusions from HULC (1927 vs 1930)and WT mice. Figure includes data for two mice for each mouse type(“mouse 1” and “mouse 2”).

FIGS. 26A-26C show sensorgrams from surface plasmon resonance bindingexperiments in which monoclonal antibodies (AA, BB, CC, DD, HH, GG, NN,and OO) from either heterozygous HULC or WT mice were allowed toassociate with the immunogen at neutral pH (pH 7.4) followed by a shiftto a buffer with pH of either 7.4 or 6.0 for the dissociation phase. Theindividual lines in each graph represent the binding responses atdifferent concentrations of the respective antibodies. All experimentswere carried out at 25° C. Dissociative half-life values (t½) are notedabove the respective sensorgrams, and fold change in t½ is included tothe right of each sensorgram. Antibodies AA, BB, CC, DD, HH, and GG werefrom HULC 1927 mice using His-substituted light chain, NN is from HULC1927 mouse using WT light chain, and 00 is from a WT mouse (See Table 5for clarification).

FIG. 27 shows positions of histidine residues engineered in the CDR3region of human Vκ3-20-derived light chains by mutagenesis. Histidineresidues introduced through mutagenesis and corresponding nucleic acidresidues are shown in bold. Amino acid positions (105, 106, etc.) arebased on a unique numbering described in Lefranc et al. (2003) Dev.Comp. Immunol. 27:55-77, and can also be viewed on www.imgt.org.

FIG. 28 shows the sequence and properties (% GC content, N, % mismatch,Tm) of selected mutagenesis primers used to engineer histidine residuesinto CDR3 of a rearranged human Vκ3-20/Jκ1 light chain sequence. SEQ IDNOs for these primers used in the Sequence Listing are included in theTable below. F=forward primer, R=reverse primer.

FIGS. 29A-29B show a general strategy for construction of targetingvectors for the engineering of histidine residues into a rearrangedhuman light chain variable region sequence derived from Vκ3-20/Jκ1 lightchain variable region for making a genetically modified mouse thatexpresses antibodies containing the modified human light chain. FIG. 29Cshows introduction of the targeting vector forULC-Q105H/Q106H/Y107H/S109H substitutions into ES cells and generationof heterozygous mice from the same; while FIG. 29D shows introduction ofthe targeting vector for ULC-Q105H/Q106H/S109H substitutions into EScells and generation of heterozygous mice from the same. The diagramsare not presented to scale. Unless indicated otherwise, filled shapesand solid lines represent mouse sequence, empty shapes and double linesrepresent human sequence.

DETAILED DESCRIPTION

This invention is not limited to particular methods, and experimentalconditions described, as such methods and conditions may vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only, and is not intendedto be limiting, since the scope of the present invention is defined bythe claims.

Unless defined otherwise, all terms and phrases used herein include themeanings that the terms and phrases have attained in the art, unless thecontrary is clearly indicated or clearly apparent from the context inwhich the term or phrase is used. Although any methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, particular methods andmaterials are now described. All publications mentioned are herebyincorporated by reference.

DEFINITIONS

The term “antibody”, as used herein, includes immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds. Each heavy chaincomprises a heavy chain variable domain and a heavy chain constantregion (C_(H)). The heavy chain constant region comprises three domains,C_(H)1, C_(H)2 and C_(H)3. Each light chain comprises a light chainvariable domain and a light chain constant region (C_(L)). The heavychain and light chain variable domains can be further subdivided intoregions of hypervariability, termed complementarity determining regions(CDR), interspersed with regions that are more conserved, termedframework regions (FR). Each heavy and light chain variable domaincomprises three CDRs and four FRs, arranged from amino-terminus tocarboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 andHCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3.The term “high affinity” antibody refers to an antibody that has a K_(D)with respect to its target epitope about of 10⁻⁹ M or lower (e.g., about1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, or about 1×10⁻¹² M). In one embodiment,K_(D) is measured by surface plasmon resonance, e.g., BIACORE™; inanother embodiment, K_(D) is measured by ELISA.

The phrase “bispecific antibody” includes an antibody capable ofselectively binding two or more epitopes. Bispecific antibodiesgenerally comprise two nonidentical heavy chains, with each heavy chainspecifically binding a different epitope—either on two differentmolecules (e.g., different epitopes on two different immunogens) or onthe same molecule (e.g., different epitopes on the same immunogen). If abispecific antibody is capable of selectively binding two differentepitopes (a first epitope and a second epitope), the affinity of thefirst heavy chain for the first epitope will generally be at least oneto two or three or four or more orders of magnitude lower than theaffinity of the first heavy chain for the second epitope, and viceversa. Epitopes specifically bound by the bispecific antibody can be onthe same or a different target (e.g., on the same or a differentprotein). Exemplary bispecific antibodies include those with a firstheavy chain specific for a tumor antigen and a second heavy chainspecific for a cytotoxic marker, e.g., an Fc receptor (e.g., FcγRI,FcγRII, FcγRIII, etc.) or a T cell marker (e.g., CD3, CD28, etc.).Further, the second heavy chain variable domain can be substituted witha heavy chain variable domain having a different desired specificity.For example, a bispecific antibody with a first heavy chain specific fora tumor antigen and a second heavy chain specific for a toxin can bepaired so as to deliver a toxin (e.g., saporin, vinca alkaloid, etc.) toa tumor cell. Other exemplary bispecific antibodies include those with afirst heavy chain specific for an activating receptor (e.g., B cellreceptor, FcγRI, FcγRIIA, FcγRIIIA, FcαRI, T cell receptor, etc.) and asecond heavy chain specific for an inhibitory receptor (e.g., FcγRIIB,CD5, CD22, CD72, CD300a, etc.). Such bispecific antibodies can beconstructed for therapeutic conditions associated with cell activation(e.g. allergy and asthma). Bispecific antibodies can be made, forexample, by combining heavy chains that recognize different epitopes ofthe same immunogen. For example, nucleic acid sequences encoding heavychain variable sequences that recognize different epitopes of the sameimmunogen can be fused to nucleic acid sequences encoding the same ordifferent heavy chain constant regions, and such sequences can beexpressed in a cell that expresses an immunoglobulin light chain. Atypical bispecific antibody has two heavy chains each having three heavychain CDRs, followed by (N-terminal to C-terminal) a C_(H)1 domain, ahinge, a C_(H)2 domain, and a C_(H)3 domain, and an immunoglobulin lightchain that either does not confer epitope-binding specificity but thatcan associate with each heavy chain, or that can associate with eachheavy chain and that can bind one or more of the epitopes bound by theheavy chain epitope-binding regions, or that can associate with eachheavy chain and enable binding of one or both of the heavy chains to oneor both epitopes.

The term “cell” includes any cell that is suitable for expressing arecombinant nucleic acid sequence. Cells include those of prokaryotesand eukaryotes (single-cell or multiple-cell), bacterial cells (e.g.,strains of E. coli, Bacillus spp., Streptomyces spp., etc.),mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S.pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells(e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni,etc.), non-human animal cells, human cells, or cell fusions such as, forexample, hybridomas or quadromas. In some embodiments, the cell is ahuman, monkey, ape, hamster, rat, or mouse cell. In some embodiments,the cell is eukaryotic and is selected from the following cells: CHO(e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cell,Vero, CV1, kidney (e.g., HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK),HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60, (e.g., BHK21),Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cell, C127 cell,SP2/0, NS-0, MMT 060562, Sertoli cell, BRL 3A cell, HT1080 cell, myelomacell, tumor cell, and a cell line derived from an aforementioned cell.In some embodiments, the cell comprises one or more viral genes, e.g., aretinal cell that expresses a viral gene (e.g., a PER.C6™ cell).

The term “complementary determining region” or “CDR,” as used herein,includes an amino acid sequence encoded by a nucleic acid sequence of anorganism's immunoglobulin genes that normally (i.e., in a wild typeanimal) appears between two framework regions in a variable region of alight or a heavy chain of an immunoglobulin molecule (e.g., an antibodyor a T cell receptor). A CDR can be encoded by, for example, a germlinesequence or a rearranged sequence, and, for example, by a naïve or amature B cell or a T cell. A CDR can be somatically mutated (e.g., varyfrom a sequence encoded in an animal's germline), humanized, and/ormodified with amino acid substitutions, additions, or deletions. In somecircumstances (e.g., for a CDR3), CDRs can be encoded by two or moresequences (e.g., germline sequences) that are not contiguous (e.g., inan unrearranged nucleic acid sequence) but are contiguous in a B cellnucleic acid sequence, e.g., as a result of splicing or connecting thesequences (e.g., V-D-J recombination to form a heavy chain CDR3).

The term “conservative,” when used to describe a conservative amino acidsubstitution, includes substitution of an amino acid residue by anotheramino acid residue having a side chain R group with similar chemicalproperties (e.g., charge or hydrophobicity). In general, a conservativeamino acid substitution will not substantially change the functionalproperties of interest of a protein, for example, the ability of avariable region to specifically bind a target epitope with a desiredaffinity. Examples of groups of amino acids that have side chains withsimilar chemical properties include aliphatic side chains such asglycine, alanine, valine, leucine, and isoleucine; aliphatic-hydroxylside chains such as serine and threonine; amide-containing side chainssuch as asparagine and glutamine; aromatic side chains such asphenylalanine, tyrosine, and tryptophan; basic side chains such aslysine, arginine, and histidine; acidic side chains such as asparticacid and glutamic acid; and, sulfur-containing side chains such ascysteine and methionine. Conservative amino acids substitution groupsinclude, for example, valine/leucine/isoleucine, phenylalanine/tyrosine,lysine/arginine, alanine/valine, glutamate/aspartate, andasparagine/glutamine. In some embodiments, a conservative amino acidsubstitution can be substitution of any native residue in a protein withalanine, as used in, for example, alanine scanning mutagenesis. In someembodiments, a conservative substitution is made that has a positivevalue in the PAM250 log-likelihood matrix disclosed in Gonnet et al.(1992) Exhaustive Matching of the Entire Protein Sequence Database,Science 256:1443-45, hereby incorporated by reference. In someembodiments, the substitution is a moderately conservative substitutionwherein the substitution has a nonnegative value in the PAM250log-likelihood matrix.

In some embodiments, residue positions in an immunoglobulin light chainor heavy chain differ by one or more conservative amino acidsubstitutions. In some embodiments, residue positions in animmunoglobulin light chain or functional fragment thereof (e.g., afragment that allows expression and secretion from, e.g., a B cell) arenot identical to a light chain whose amino acid sequence is listedherein, but differs by one or more conservative amino acidsubstitutions.

The term “dissociative half-life” or “t_(1/2)” as used herein refers tothe value calculated by the following formula: t_(1/2) (min)=(ln2/k_(d))/60, wherein k_(d) represents a dissociation rate constant.

The phrase “epitope-binding protein” includes a protein having at leastone CDR and that is capable of selectively recognizing an epitope, e.g.,is capable of binding an epitope with a K_(D) that is at about onemicromolar or lower (e.g., a K_(D) that is about 1×10⁻⁶ M, 1×10⁻⁷ M,1×10⁻⁹ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, or about 1×10⁻¹² M).Therapeutic epitope-binding proteins (e.g., therapeutic antibodies)frequently require a K_(D) that is in the nanomolar or the picomolarrange.

The term “functional” as used herein, e.g., in reference to a functionalpolypeptide, includes a polypeptide that retains at least one biologicalactivity normally associated with the native protein. In anotherinstance, a functional immunoglobulin gene segment may include avariable gene segment that is capable of productive rearrangement togenerate a rearranged immunoglobulin gene sequence.

The phrase “functional fragment” includes fragments of epitope-bindingproteins that can be expressed, secreted, and specifically bind to anepitope with a K_(D) in the micromolar, nanomolar, or picomolar range.Specific recognition includes having a K_(D) that is at least in themicromolar range, the nanomolar range, or the picomolar range.

The term “germline” as used herein, in reference to an immunoglobulinnucleic acid sequence, includes reference to nucleic acid sequences thatcan be passed to progeny.

The phrase “heavy chain,” or “immunoglobulin heavy chain” includes animmunoglobulin heavy chain sequence, including immunoglobulin heavychain constant region sequence, from any organism. Heavy chain variabledomains include three heavy chain CDRs and four FR regions, unlessotherwise specified. Fragments of heavy chains include CDRs, CDRs andFRs, and combinations thereof. A typical heavy chain has, following thevariable domain (from N-terminal to C-terminal), a C_(H)1 domain, ahinge, a C_(H)2 domain, and a C_(H)3 domain. A functional fragment of aheavy chain includes a fragment that is capable of specificallyrecognizing an epitope (e.g., recognizing the epitope with a K_(D) inthe micromolar, nanomolar, or picomolar range), that is capable ofexpressing and secreting from a cell, and that comprises at least oneCDR. Heavy chain variable domains are encoded by variable regionnucleotide sequence, which generally comprises V_(H), D_(H), and J_(H)segments derived from a repertoire of V_(H), D_(H), and J_(H) segmentspresent in the germline. Sequences, locations and nomenclature for V, D,and J heavy chain segments for various organisms can be found in IMGTdatabase, which is accessible via the internet on the world wide web(www) at the URL “imgt.org.”

The term “identity” when used in connection with sequence, includesidentity as determined by a number of different algorithms known in theart that can be used to measure nucleotide and/or amino acid sequenceidentity. In some embodiments described herein, identities aredetermined using a ClustalW v. 1.83 (slow) alignment employing an opengap penalty of 10.0, an extend gap penalty of 0.1, and using a Gonnetsimilarity matrix (MACVECTOR™ 10.0.2, MacVector Inc., 2008). The lengthof the sequences compared with respect to identity of sequences willdepend upon the particular sequences, but in the case of a light chainconstant domain, the length should contain sequence of sufficient lengthto fold into a light chain constant domain that is capable ofself-association to form a canonical light chain constant domain, e.g.,capable of forming two beta sheets comprising beta strands and capableof interacting with at least one C_(H)1 domain of a human or a mouse. Inthe case of a C_(H)1 domain, the length of sequence should containsequence of sufficient length to fold into a C_(H)1 domain that iscapable of forming two beta sheets comprising beta strands and capableof interacting with at least one light chain constant domain of a mouseor a human.

The phrase “immunoglobulin molecule” includes two immunoglobulin heavychains and two immunoglobulin light chains. The heavy chains may beidentical or different, and the light chains may be identical ordifferent.

The phrase “light chain” includes an immunoglobulin light chain sequencefrom any organism, and unless otherwise specified includes human kappa(K) and lambda (A) light chains and a VpreB, as well as surrogate lightchains. Light chain variable domains typically include three light chainCDRs and four framework (FR) regions, unless otherwise specified.Generally, a full-length light chain includes, from amino terminus tocarboxyl terminus, a variable domain that includesFR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, and a light chain constant region aminoacid sequence. Light chain variable domains are encoded by the lightchain variable region nucleotide sequence, which generally compriseslight chain V_(L) and light chain J_(L) gene segments, derived from arepertoire of light chain V and J gene segments present in the germline.Sequences, locations and nomenclature for light chain V and J genesegments for various organisms can be found in IMGT database, which isaccessible via the internet on the world wide web (www) at the URL“imgt.org.” Light chains include those, e.g., that do not selectivelybind either a first or a second epitope selectively bound by theepitope-binding protein in which they appear. Light chains also includethose that bind and recognize, or assist the heavy chain with bindingand recognizing, one or more epitopes selectively bound by theepitope-binding protein in which they appear. Light chains also includethose that bind and recognize, or assist the heavy chain with bindingand recognizing, one or more epitopes selectively bound by theepitope-binding protein in which they appear. Common or universal lightchains include those derived from a human Vκ1-39Jκ5 gene or a humanVκ3-20Jκ1 gene, and include somatically mutated (e.g., affinity matured)versions of the same.

The phrase “micromolar range” is intended to mean 1-999 micromolar; thephrase “nanomolar range” is intended to mean 1-999 nanomolar; the phrase“picomolar range” is intended to mean 1-999 picomolar.

“Neutral pH” includes pH between about 7.0 and about 8.0, e.g., pHbetween about 7.0 and about 7.4, e.g., between about 7.2 and about 7.4,e.g., physiological pH in, e.g., a mouse or a human. “Acidic pH”includes pH of 6.0 or lower, e.g., pH between about 5.0 and about 6.0,pH between about 5.75 and about 6.0, e.g., pH of endosomal or lysosomalcompartments.

The term “operably linked” refers to a relationship wherein thecomponents operably linked function in their intended manner. In oneinstance, a nucleic acid sequence encoding a protein may be operablylinked to regulatory sequences (e.g., promoter, enhancer, silencersequence, etc.) so as to retain proper transcriptional regulation. Inone instance, a nucleic acid sequence of an immunoglobulin variableregion (or V(D)J segments) may be operably linked to a nucleic acidsequence of an immunoglobulin constant region so as to allow properrecombination between the sequences into an immunoglobulin heavy orlight chain sequence.

The phrase “somatically mutated,” as used herein, includes reference toa nucleic acid sequence from a B cell that has undergoneclass-switching, wherein the nucleic acid sequence of an immunoglobulinvariable region, e.g., a heavy chain variable region (e.g., a heavychain variable domain or including a heavy chain CDR or FR sequence) inthe class-switched B cell is not identical to the nucleic acid sequencein the B cell prior to class-switching, such as, for example adifference in a CDR or a framework nucleic acid sequence between a Bcell that has not undergone class-switching and a B cell that hasundergone class-switching. The phrase “somatically mutated” includesreference to nucleic acid sequences from affinity-matured B cells thatare not identical to corresponding immunoglobulin variable regionnucleotide sequences in B cells that are not affinity-matured (i.e.,sequences in the genome of germline cells). The phrase “somaticallymatured” also includes reference to an immunoglobulin variable regionnucleic acid sequence from a B cell after exposure of the B cell to anepitope of interest, wherein the nucleic acid sequence differs from thecorresponding nucleic acid sequence prior to exposure of the B cell tothe epitope of interest. The term “somatically mutated” also refers tosequences from antibodies that have been generated in an animal, e.g., amouse having human immunoglobulin variable region nucleic acidsequences, in response to an immunogen challenge, and that result fromthe selection processes inherently operative in such an animal.

The term “unrearranged,” with reference to a nucleic acid sequence,includes nucleic acid sequences that exist in the germline of an animalcell.

The phrase “variable domain” includes an amino acid sequence of animmunoglobulin light or heavy chain (modified as desired) that comprisesthe following amino acid regions, in sequence from N-terminal toC-terminal (unless otherwise indicated): FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The phrase “operably linked” refers to a juxtaposition wherein thecomponents so described are in a relationship permitting them tofunction in their intended manner. In one instance, a nucleic acidsequence encoding a protein may be operably linked to regulatorysequences (e.g., promoter, enhancer, silencer sequence, etc.) so as toretain proper transcriptional regulation. In one instance, a nucleicacid sequence of an immunoglobulin variable region (or V(D)J segments)may be operably linked to a nucleic acid sequence of an immunoglobulinconstant region so as to allow proper recombination between thesequences into an immunoglobulin heavy or light chain sequence.

The term “replacement” in reference to gene replacement refers toplacing exogenous genetic material at an endogenous genetic locus,thereby replacing all or a portion of the endogenous gene with anorthologous or homologous nucleic acid sequence.

The term “functional” as used herein, e.g., in reference to a functionalpolypeptide, includes a polypeptide that retains at least one biologicalactivity normally associated with the native protein. In anotherinstance, a functional immunoglobulin gene segment may include avariable gene segment that is capable of productive rearrangement togenerate a rearranged immunoglobulin gene sequence.

Variable Domains with Histidine Substitutions

The design of human immunoglobulin-based therapeutics is a well-studiedphenomenon, yet certain unsolved problems persist in making suchtherapeutics with optimal characteristics, e.g., extending serumhalf-life of such therapeutics or otherwise improving their ability tobind more target per therapeutic molecule. Much work over the lastcouple of decades aimed at elucidating serum immunoglobulin turnover hasfocused on ways to increase serum half-life of therapeutically importantantibodies, or immunoglobulin-based therapeutics, by modifying antibodystructure. For the most part, this modification work has focused on theinteraction of the constant domains of antibodies with the neonatal Fcreceptor (FcRn). The neonatal Fc receptor on the extracellular surfacebinds circulating antibodies through their Fc regions to form anantibody-FcRn complex that is incorporated, or endocytosed, into thecell where the ligand and antibody part ways and the antibody-FcRncomplex undergoes a cycling process that brings the antibody and theFcRn back to the cell's surface where the antibody is released and canre-bind a new target molecule. Cycling of the antibody-FcRn complexbecame an area of intense interest following the discovery of generalmechanisms of receptor cycling.

Receptor cycling can proceed by a variety of mechanisms.Receptor-mediated endocytosis provides an endosomal pathway for aregulated recycling of cell surface receptors and (in some cases, e.g.,FcRns) their ligands. (Pinocytosed molecules are otherwise typicallyshuttled through an endosomal pathway that ends in degradation.) Thediscovery of the mechanism of receptor-mediated endocytosis and the bodyof work concerning recycling of membrane receptors provided a frameworkfor a detailed understanding of receptor-ligand turnover in general (fora review see, e.g., Brown, M. S., Anderson, R. G. W., and Goldstein, J.L. (1983) Recycling Receptors: The Round-Trip Itinerary of MigrantMembrane Proteins, Cell 32:663-667; see also, Goldstein, J. L. andBrown, M. S. (2009) The LDL Receptor, Arterioscler. Thromb. Vasc. Biol.29:431-438; Basu, S. K. (1984) Receptor-mediated endocytosis: Anoverview of a dynamic process, J. Biosci. 6(4):535-542). Other work onendosomal sorting helped properly frame the question of the fate ofcirculating immunoglobulins and the phenomenon of immunoglobulinreceptor recycling and pharmacokinetics of antibody drugs. This workrevealed a complex antibody-FcRn complex cycling process that appears tobe primarily responsible for the relatively long half-life of IgGmolecules in serum. Indeed, even rather early work in this areaestablished that endosomes are the most plentiful in vivo source of FcRn(see, Roberts, D. M. et al. (1990) Isolation and Characterization of theFc Receptor from the Fetal Yolk Sac of the Rat, J. Cell. Biol.111:1867-1876). And it had long been observed that receptor-positiveendosomal fractions are in large part not headed for lysosomaldegradation (see, e.g., Brown, M. S. et al. (1983) Recycling Receptors:The Round-Trip Itinerary of Migrant Membrane Proteins, Cell 32:663-667;see also, von Figura et al. (1984) Antibody to mannos 6-phosphatespecific receptor induces receptor deficiency in human fibroblasts, EMBOJ. 3(6):1281-1286), in the absence of aggregation (see, e.g., Dunn, K.W. et al. (1989) Iterative Fractionation of Recycling Receptors fromLysosomally Destined Ligands in an Early Sorting Endosome, J. Cell.Biol. 109(6):3303-3314). It is this endosomal system that participatesin a cycling process that ensures that antibodies that bind FcRn wellunder acidic conditions (e.g., human IgG1 antibodies) persist for anextended period of time in serum.

According to some reports, the recycling mechanism of FcRn-containingendosomes is novel and unusual; it does not involve ubiquitin-dependentcomplete organelle merging but rather resembles incomplete mergingmediated by tubular extensions more similar to a kiss-and-linger model(Gan, Z. et al. (2009) Analyses of the recycling receptor, FcRn, in livecells reveal novel pathways for lysosomal delivery, Traffic 10(5):600;see also, Tzaban, S. et al. (2009) The recycling and transcytoticpathways for IgG transport by FcRn are distinct and display an inherentpolarity, J. Cell Biol. 185(4):673-684). Thus, the antibody-FcRn cyclingmodel appears to be distinct from other endosomal pathways.

The antibody-FcRn endosomal cycling mechanism assures that antibodiesthat bind well to FcRn are able to sustain prolonged presence in serumthrough a more or less continuous FcRn-protective process that entailssequestering bound antibody in an endosomal compartment where thebinding of antibody to FcRn is maintained, preventing lysosomaldegradation of antibody bound to FcRn. Typically, circulating antibodymolecules bind FcRn on the cell surface. Antibody-FcRn complexes appearin endosomes as the result of a continuous endocytosis process.FcRn-bound molecules (e.g., antibodies, or Fc fusion proteins) remainassociated with FcRn in the acidic endosomal compartment throughacid-stable Fc-FcRn interaction. Molecules not bound to the endosomalsurface (through, e.g., FcRn or another receptor) are shuttled to thelysosomal pathway and degraded, whereas receptor-bound molecules arerecycled to the plasma membrane when the endosome fuses with the plasmamembrane. Upon fusion with the plasma membrane, the acid-stable Fc-FcRninteraction is exposed to a near neutral extracellular pH where the Fcreadily dissociates from the FcRn. It is the pH binding differential ofthe Fc, coupled with a differential thermal stability of FcRn thatvaries with protonation state, that is believed to be primarilyresponsible for the ability of certain Fc's to sustain serumconcentrations through binding FcRn. A key to the endosomal cyclingmechanism is ligand release by receptors in the acidic endosomalcompartment (reviewed, e.g., in Brown, M. S. et al. (1983)).

IgG1 Fc moieties bind FcRn with high affinity at pH 6.0 to about 6.5;binding at pH 7.0 to about pH 7.5 is about two orders of magnitudeweaker, presumably due to titration of histidine residues near theregion of the Fc that binds FcRn, residues 310-433, and an FcRnintramolecular thermal stability differential mediated by protonationstate (Raghaven, M. et al. (1995) Analysis of the pH dependence of theneonatal Fc receptor/immunoglobulin G interaction using antibody andreceptor variants, Biochemistry 34:14649-14657; Vaughn, D. E. andBjorkman, P. J. (1998) Structural basis of pH-dependent antibody bindingby the neonatal Fc receptor, Structure 6:63-73); it has beendemonstrated that rat FcRn exhibits a better thermal stability profileat pH 6.0 than at pH 8.0 (Raghavan, M. et al. (1993) The class IMHC-related Fc receptor shows pH dependent stability differencescorrelating with immunoglobulin binding and release, Biochemistry32:8654-8660).

Although nature gave rise to Fc structures that bind FcRndifferentially, the science of Fc engineering arose to design Fcstructures that would result in tighter binding to FcRnand—presumably—longer serum half-life. Many such structures weredesigned and tested, far too numerous to review here, with varyingdegrees of success. Mutating immunoglobulin constant region sequences topromote recycling of antibody by modifying FcRn binding characteristicshas a long and varied history. To date, most if not all effort toidentify mutations have focused on residues believed to be critical inbinding or interacting with FcRn, i.e., residues whose modificationaffect affinity of the Fc for FcRn.

But binding of Fc to FcRn is itself a complex matter. Different types oftherapeutic antibodies (humanized, chimeric, and mouse), and even withintypes (e.g., comparing different humanized antibodies to one another,comparing IgG1 isotype antibodies to one another, etc.), exhibitdissociation constants with respect to FcRn that vary as much as abouttwo-fold (see, e.g., Suzuki, T. et al. (2010) Importance of Neonatal FcRin Regulating the Serum Half-Life of Therapeutic Proteins Containing theFc Domain of Human IgG1: A Comparative Study of the Affinity ofMonoclonal Antibodies and Fc-Fusion Proteins to Human Neonatal FcR, J.Immunol. 184:1968-1976). This observation permits an inference that theprimary structure of the constant region may not account for allpharmacokinetic behavior. Others have postulated that overallisoelectric point (pi) of an antibody, keeping the constant regionprimary structure fixed, is an important determinant of serumhalf-life—presumably through an unspecified non-FcRn-dependent mechanism(Igawa, T. et al. (2010) Reduced elimination of IgG antibodies byengineering the variable region, Protein Engineering, Design & Selection23(5):385-392). Under this view, the lower the pl of the antibody, thetighter the binding to FcRn (Id.). For at least one IgG4 isotypeantibody, a change in pl from 9.2 to 7.2 correlated with a 2.4-foldincrease in half-life and a 4.4-fold reduction in clearance (Id.),consistent with an inference that a nonspecific lowering of pl bymodifying residues in both the heavy and light chain variable regionstogether can significantly impact pharmacokinetic behavior. In thatreport, residue modification did not follow any particular pattern andno residue was substituted to histidine, although at least one residuein a light chain CDR2 was changed from a histidine residue to aglutamate residue (Id., at FIG. 5, p. 390). Further, an odd paradox mayerupt when comparing in vitro FcRn binding and in vivo pharmacokinetics:for at least one clinically important IgG1 antibody with multiplesubstitutions in the Fc region that interacts with FcRn, in vitro FcRnbinding did not correlate with in vivo pharmacokinetic behavior (see,Petkova, S. B. et al. (2006) Enhanced half-life of geneticallyengineered human IgG1 antibodies in a humanized FcRn mouse model:potential application in humorally mediated autoimmune disease, IntlImmunol. 18(12):1759-1769). Finally, release of Fc ligand from FcRn uponfusion with the plasma membrane appears to occur in two phases—a rapidphase and an extended phase—of unknown mechanism (see, Ober R. J. et al.(2004) Exocytosis of IgG as mediated by the receptor FcRn: an analysisat the single-molecule level, Proc. Natl. Acad. Sci. USA101:11076-11081).

Finally, extending half-life of antibodies in serum is one way toenhance efficiency of antibody therapy. Improved efficacy, or improvedavailability of the same antibody or variable domain to bind andeliminate two or three or more target molecules are not necessarilyaddressed by improving FcRn binding and turnover that affects targetantigen. Modifications that increase affinity of an Fc to FcRN areexpected to increase turnover and thus improve pharmacokinetics of atherapeutic antibody. Antigen-antibody complexes bind FcRn tightly,resulting in the antigen-antibody complex cycling back intoextracellular space rather than being degraded by a lysosomal pathway.In this scenario, however, the antigen, or target, may largely remaincomplexed to the antibody and recycled together with the antibody intothe extracellular space. For therapeutic antibodies, this phenomenon canbe very undesirable.

However, antibodies whose interaction with antigen are pH-dependent,i.e., antibodies engineered to bind antigen with lower affinity at anendosomal pH, would not recycle antigen in an FcRn-dependent manner dueto instability of the antigen-antibody complex in the endosomalcompartment. This is because in the acidic environment of the endosome,the antigen will disengage from the antibody-FcRn complex, and theantibody-bound FcRn will recycle to the surface of the cell, whereasdisengaged free antigen will shuttle to a lysosomal degradation pathway.In this way, pH-dependent antigen binding can provide enhanced efficacyand/or pharmacokinetics within the context of FcRn-mediated cycling (butnot directly depending on the Fc-FcRn interaction) by freeing cycledantibody to bind antigen, bind FcRn, cycle through endosomes, andre-enter the extracellular space to bind more antigen and shuttle moreantigen to a lysosomal degradation pathway.

Capitalizing on the observation that ligands will frequently dissociatefrom their receptors at an endosomal pH, it had been suggested to searchfor antibodies that effectively release antigen at an endosomal pH inorder to make certain specific multifunctional molecules that targetspecific cells in order to import toxins into the cells and release thetoxins within endosomes (see, e.g., U.S. Pat. Nos. 5,599,908 and5,603,931). But that does not address antigen-antibody cycling, inparticular for human therapeutics.

To leverage antibody structure for shuttling endosomally disengagedantigen through a lysosomal pathway while maintaining FcRn-dependentcycling of antigen-FcRn complexes, certain approaches to pH-dependentantigen binding have been explored. Such approaches include ageneralized histidine-scanning over the variable region to substituteresidues with histidine and test to see whether the generalized approachof histidine replacement yields an antibody with desired pH-dependentantigen binding (see, e.g., Igawa, T. et al. (2010) Antibody recyclingby engineered pH-dependent antigen binding improves the duration ofantigen neutralization, Nature Biotech. 28(11):1203-1208; see also, U.S.Patent Application Publication No. 2011/0111406 A1)). A likelydisadvantage of this approach is that modifying residues important forantigen binding are likely to disrupt binding at either an acidic orneutral pH, which can eliminate any leverage due to the pH differentialbetween the endosomal compartment and the extracellular space.

In various aspects, compositions and methods are provided for making oneor more histidine substitutions at a few judiciously-selected regions inan antibody variable region (heavy chain and/or light chain variabledomain) provides a method for making antibody variable domains that binda target antigen in a pH-dependent manner, e.g., variable domains thatbind an antigen of interest with a first affinity at a neutral or basicor extracellular pH, yet bind the same antigen of interest with a secondaffinity at an acidic pH, wherein the first affinity is high and whereinthe second affinity is low.

In various aspects, the one or more histidine substitutions are in aCDR1, a CDR2, a CDR3, an N-terminal, and/or a loop 4 sequence.

In some aspects the one or more histidine substitutions are in a CDR1, aCDR2, and/or a CDR3.

In some aspects, the one or more histidine substitutions are in a CDR3and a loop 4 sequence. In a further embodiment, the substitutions arealso in an N-terminal sequence.

In some aspects, the one or more histidine substitutions are in a CDR3and an N-terminal sequence. In a further embodiment, the substitutionsare also in a loop 4 sequence.

In some aspects, the one or more histidine substitutions are in a CDR2sequence and a loop 4 sequence. In a further embodiment, thesubstitutions are also in an N-terminal sequence.

In some aspects, the loop 4 sequence is for a λ light chain variabledomain residues 83-88; for a κ light chain variable domain residues83-88; and for a heavy chain variable region 82-88 (IMGT numbering).

In some aspects, the N-terminal sequence for a light chain variabledomain or a heavy chain variable domain are residues 1-26 (IMGTnumbering). In one embodiment, the N-terminal sequence that comprisesone or more (e.g., clustered) histidine substitutions is residues 1-5,in one embodiment residues 1-10, in one embodiment 1-15, in oneembodiment 1-20, in one embodiment 1-25, in one embodiment 5-10, in oneembodiment 10-15, in one embodiment 15-20, in one embodiment 20-25, inone embodiment 5-15, in one embodiment 10-20, in one embodiment 5-20. Inone embodiment, the histidine substitutions are two or more (e.g.,three, four, five, or six or more), and at least two or more of thehistidine substitutions are made within a stretch of N-terminal sequencethat is about 3 residues, 4 residues, five residues, or six residues ormore. In one embodiment, a plurality of histidine substitutions are madein the N-terminal, and the histidine substitutions comprise clusters ofat least two, at least three, or at least four histidine substitutions.In one embodiment, at least one cluster of histidine substitutionscomprises histidine substitutions that are separated by one or morenon-histidine substitutions.

In some aspects, the one or more histidine substitutions the CDR aretwo, three, four, five, or six substitutions within the CDR. In oneembodiment, all residues in the CDR that are not critical for binding ata neutral pH are substituted with a histidine. In one embodiment, thetwo, three, four, five, or six substitutions are contiguous; in oneembodiment, one or more of the two, three, four, five, or sixsubstitutions are present in a cluster, wherein the cluster comprises atleast one non-histidine residue; in one embodiment, the clustercomprises two non-histidine residues; in one embodiment, the clustercomprises three non-histidine residues; in one embodiment, the clustercomprises four non-histidine residues.

In some aspects, the one or more histidine substitutions in theN-terminal are one, two, three, four, five, or six substitutions. In oneembodiment, all residues in the N-terminal that do not reduce antigenbinding at a neutral pH (e.g., by more than 1%, 2%, 3%, 4%, or 5%), aresubstituted with a histidine. In one embodiment, the two, three, four,five, or six substitutions are contiguous; in one embodiment, one ormore of the two, three, four, five, or six substitutions are present ina cluster, wherein the cluster comprises at least one non-histidineresidue; in one embodiment, the cluster comprises two non-histidineresidues; in one embodiment, the cluster comprises three non-histidineresidues; in one embodiment, the cluster comprises four non-histidineresidues.

In some aspects, the method comprises modifying a variable domain tocomprise a cluster of histidine substitutions (e.g., as describedherein, contiguous or interrupted with one or more non-histidineresidues) in a region selected from a CDR1, a CDR2, a CDR3, anN-terminal, a loop 4, and a combination thereof. In some aspects, thecluster is a sequence bounded upstream by a first histidine residue, anddownstream by a second histidine residue, and comprises one or moreresidues between the first and second histidine residues. In oneembodiment, the one or more residues between the first and secondhistidine residues are 1, 2, 3, 4, 5, or 6 or more non-histidineresidues. In one embodiment, the one or more residues between the firstand second histidine residues are 1, 2, 3, 4, 5, or six histidineresidues. In one embodiment, the cluster is 3 residues, in oneembodiment 4 residues, in one embodiment 5 residues, in one embodiment 6residues, in one embodiment 7 residues, in one embodiment 8 residues ormore.

In various aspects, the method comprises identifying sequences in anantibody variable domain (heavy and/or light chain) that are criticalfor binding antigen (e.g., at a neutral pH, e.g., pH 7-7.4, e.g., pH7.2, e.g., an extracellular pH), and substituting one or more residueswithin the sequence to histidine, wherein the substitution to histidinedoes not eliminate binding of the variable domain to a target antigen ata neutral pH. In various aspects, a cluster of two or more, three ormore, four or more, or five or more residues that are not critical forbinding at a neutral pH are substituted with histidine residues. Invarious aspects, the cluster of histidine residues is within a CDR, aloop 4, an N-terminal, or a combination thereof.

In various aspects, a residue that is critical for binding is identifiedas a residue that when substituted with a substitute amino acid at abouta neutral (or extracellular) pH, reduces binding of the variable domainby in one embodiment at least 5%, in one embodiment at least 10%, in oneembodiment at least 20%, in one embodiment at least 30%, in oneembodiment at least 40%, in one embodiment at least 50%, in oneembodiment at least 60%, in one embodiment at least 70%, in oneembodiment at least 80%, in one embodiment at least 90%, in oneembodiment results in no detectable binding. In one embodiment, thesubstitute amino acid is a histidine. In one embodiment, the substituteamino acid is an alanine.

In one aspect, a method is provided for making an antibody variabledomain that binds and antigen weaker at an acidic pH than it binds thesame antigen at a neutral or basic pH, wherein the method comprisessubstituting one or more amino acid residues of the variable region withone or more histidine residues. In one embodiment, the binding at theacidic pH is negligible or zero.

In one embodiment, the one or more amino acid residues substituted arein a light chain. In a specific embodiment, the one or more residues arein an N-terminal region of a light chain. In a specific embodiment, theN-terminal residues are selected from 1-26, 1-20, 1-15, 1-10, 1-6, or1-5 (IMGT numbering). In one embodiment, the one or more residues are inloop 3. In a specific embodiment, the loop 4 residues are 83-88 in Vκ orVλ, and 82-88 in V_(H) (IMGT numbering).

In one embodiment, the one or more residues are in a heavy chain. In aspecific embodiment, the one or more residues are in an N-terminalregion of a heavy chain. In a specific embodiment, the N-terminalresidues are selected from 1-26, 1-20, 1-15, 1-10, 1-6, or 1-5 (IMGTnumbering). In one embodiment, the one or more residues are in loop 4.In a specific embodiment, the loop 3 residues are selected from (for Vκand/or Vλ) 83, 84, 85, 86, 87, 88, and a combination thereof (IMGTnumbering); or (for V_(H)) 82, 83, 84, 85, 86, 87, 88, and a combinationthereof (IMGT numbering); and, a combination thereof.

In one embodiment, the one or more residues are in a CDR selected from aCDR1, a CDR2, and a CDR3; and the one or more residues when substituted(e.g., with alanine or with histidine) do not result in decreasedbinding of the target antigen at a neutral or a basic pH. In a specificembodiment, decreased binding of the target antigen at neutral or basicpH as the result of substitution (e.g., by alanine or histidinesubstitution) is no more than 5%, no more than 10%, no more than 15%, orno more than 20%, no more than 25%, or no more than 30% as compared withnon-substituted variable domain.

In some aspects, the his-modified variable domain complexed with thetarget antigen exhibits a half-life of at least about 20 minutes at anelevated pH (e.g., an extracellular pH, or a pH from 7-7.4, e.g., pH7.2) and exhibits a half-life of less than 5 minutes, less than 4minutes, less than 3 minutes, less than 2 minutes, or less than a minuteat an endosomal pH, or a pH from e.g., pH 5-6, e.g., pH 5.75. In oneembodiment, the his-modified variable domain complexed with the targetantigen exhibits a half-life of at least about 20 minutes at theelevated pH, and exhibits a half-life at an endosomal pH of less than 60seconds, less than 30 seconds, less than 10 seconds, less than 5seconds, less than 4 seconds, less than 3 seconds, or less than 2seconds. In one embodiment, the his-modified variable domain complexedwith the target antigen exhibits a half-life of at least about 20minutes at the elevated pH (e.g., pH 7-7.4, e.g., pH 7.2), and exhibitsa half-life at an endosomal pH of less than about a second, less than0.5 second, less than 0.1 second, or less than 0.05 second. In oneembodiment, half-life at an endosomal pH is measured using a BIACORE™assay in which his-modified variable domain complexed with targetantigen is equilibrated on the surface of a BIACORE™ chip at neutral orelevated pH, and buffer at an endosomal pH (or, e.g., a pH of 5-6, e.g.,pH 5.75) is flowed over the complex.

In various aspects, a method for making an antibody variable domain thatbinds a target antigen with a first affinity at an extracellular pH, andthat does not bind the target antigen or binds the target antigen with asecond affinity at an endosomal pH, wherein the first affinity is about10, 10²-, 10³-, 10⁴-, 10⁵-, 10⁶-, 10⁷-, 10⁸-, 10⁹, 10¹⁰-, 10¹¹-, or10¹²-fold (or higher-fold) than the second affinity. In some aspects,the first affinity is in the picomolar to nanomolar range (e.g., K_(D)is 10⁻¹² to 10⁻⁹), and the second affinity is in the micromolar orhigher range (e.g., K_(D)=10⁻⁶ or greater, e.g., 10⁻⁵, 10⁻⁴, 10⁻³, 10⁻²,10⁻¹, 1, or higher). In some aspects, the first affinity is in the rangeof about K_(D) 10⁻⁹ to about K_(D) 10⁻¹², and the second affinity is inthe range of about K_(D) 10⁻³ to about 1 or larger. In one embodiment,the first affinity is in the range of about K_(D) 10⁻⁹ to about K_(D)10⁻¹², and the second affinity is characterized by a K_(D)>1; in aspecific embodiment, the second affinity is characterized by a K_(D)>>1(e.g., 10, 10², 10³ or higher). In a specific embodiment, the firstaffinity is characterized by a K_(D) from about 10⁻⁹ to about 10⁻¹², andthe second affinity is characterized by an inability to detect bindingover background in a BIACORE™ binding assay.

Various aspects are illustrated by a particular case in which a humanlight chain variable sequence is modified to contain a one or more,including a cluster, of histidines in a light chain CDR3, and the lightchain is expressed in a CHO cell with a cognate human heavy chain. Theidentity of the antigen to which the histidine-modified antibody bindsis unimportant, as is the particular sequence of the light chainvariable domain. The principles illustrated in the Examples areapplicable to CDR3, CDR2, CDR1, the N-terminal region, or loop 4. Forexample, residues in the cited regions can be substituted for histidine,alone or in clusters of 2, 3, 4, or 5, e.g., and the resultingantibodies tested for pH-dependent antigen binding.

Methods for engineering antibodies that are capable of binding to anantigen in a pH dependent manner can be made by making modifications ofan immunoglobulin light chain variable region at one or more positionsalong the sequence of the light chain as described (e.g., in a CDR3,CDR2, CDR1, loop 4, N-terminal). Histidines are tolerated in CDRregions; light chains, typically show somatic hypermutation along thevariable region sequence, and, in some cases, such mutations can resultin a substitution of histidine residues in CDRs (FIG. 15).

In the Examples, histidine substitutions have been identified at one tofour positions in the light chain CDR3 region, at residues not criticalfor binding target antigen at a neutral pH, from which fifteen mutantconstructs were made. The particular light chain shown—with a variety ofdifferent but cognate heavy chains—is derived from a single Vκ and asingle Jκ segment (Vκ1-39/Jκ5). Such mutants when expressed confer uponthe antibody (in conjunction with a cognate heavy chain) the property ofpH-dependent antigen binding. The mutant constructs were made usingantigen-specific antibody variable domains and tested for expression andantigen binding at approximately a neutral pH and release at low pH(“catch-and-release”). In certain examples shown the locations of thefour identified residues (where mutation to histidine is not criticalfor binding at neutral pH) are Q105H, Q106H, Y108H and P111H. For anantibody that binds a different target antigen, or for an antibodycomprising a different rearranged V-J sequence, his-mutatable residuesfor making a pH-dependent variable domain are found by identifying whichresidues are not critical for binding at neutral pH, then modifying oneor more of those residues (e.g., in clusters) and expressing an antibodycomprising the mutations, and testing for binding (and/or release time,e.g., t_(1/2)) at a neutral pH (e.g., an extracellular pH) and at anacidic pH (e.g., an endosomal pH). Although the data shown here are fora Vκ1-39/Jκ5 light chain, other light chains, including those derivedfrom a Vκ3-20/Jκ1 rearrangement, are amenable to the approach describedherein, as are heavy chains.

All of the histidine-engineered light chain constructs that were made inthis experiment expressed well in conjunction with heavy chains.Further, binding of the antibodies to antigen in a pH-dependent mannerwas demonstrated from BIACORE™ assay data showing the binding of antigenat around a neutral pH and at an acidic pH for the 15 mutants with fivedifferent heavy chains that specifically recognize the same cell surfaceantigen (FIG. 19A-J).

The methods described, and those particular methods used for purposes ofillustration in certain of the examples and figures herein, are usefulto generate variable regions of antibodies that can be used to make,e.g., human therapeutic binding proteins that bind their targets byhuman immunoglobulin variable domains that comprise the histidines in aCDR3. The altered binding at a lower pH will in some circumstances allowfaster turnover because the therapeutic will bind a target on a cell'ssurface, be internalized in an endosome, and more readily or morerapidly dissociate from the target in the endosome, so that thetherapeutic can be recycled to bind yet another molecule of target(e.g., on another cell or the same cell). In various embodiments, thiswill result in the ability to dose the therapeutic at a lower dose, ordose the therapeutic less frequently. This is particularly useful whereit is not desirable to dose frequently, or to administer above a certaindosage, for safety or toxicity reasons. For example, the half-life of anantibody therapeutic in the serum of a subject will be increased as aresult.

Thus, in various embodiments codons in a gene encoding a rearrangedhuman light chain can be made at positions 105, 106, 108, 111, or acombination thereof. For example, position 105 in conjunction with oneor more of 106, 108, and 111; position 106 in conjunction with one ormore of 105, 108, and 111; position 108 in conjunction with one or moreof 105, 106, and 111; position 111 in conjunction with one or more of105, 106, and 108. Corresponding positions in other light chains (i.e.,derived from other V-J rearrangements) are included in variousembodiments.

Non-Human Animals that Express Immunoglobulin Heavy Chain VariableDomain Comprising Histidine Residues

The described invention provides genetically modified non-human animalsthat can produce antigen-binding proteins with pH-dependent antigenbinding characteristics. In various embodiments, the antigen-bindingproteins produced by the genetically modified non-human animals asdescribed herein exhibit increased pH-dependent recycling efficiencyand/or enhanced serum half-life. In particular, the described inventionemploys genetic modifications in the immunoglobulin heavy chain locus tointroduce histidine codons into a human heavy chain variable regionnucleotide sequence and, optionally, to introduce a mutation(s) in aconstant region nucleotide sequence that encodes C_(H)2 and/or C_(H)3domains that increases the binding of the antibody constant region to anFcRn receptor, which facilitates recycling of the antigen-bindingprotein. Antigen-binding proteins comprising the modification may moreloosely bind its target in an acidic intracellular compartment (e.g., inan endosome where pH ranges from about 5.5 to about 6.0) than in anextracellular environment or at the surface of a cell (i.e., at aphysiological pH, e.g., a pH ranging from about 7.0 to about 7.4) due toprotonated histidine residues located in the antigen binding sites.Therefore, the antigen-biding proteins comprising the geneticmodifications as described herein would be able to be recycled morerapidly or efficiently than wild-type antigen-binding proteins that donot comprise such genetic modifications following target-mediatedendocytosis. Furthermore, since the modified histidine residues areprotonated only in an acidic environment, but not at a neutral pH, it isexpected that such modification would not affect binding affinity and/orspecificity of the antigen-binding protein toward an antigen of interestat a physiological pH.

In various aspects, non-human animals are provided comprisingimmunoglobulin heavy chain loci that comprise an unrearranged humanheavy chain variable region nucleotide sequence, wherein theunrearranged human heavy chain variable region nucleotide sequencecomprises an addition of least one histidine codon or a substitution ofat least one endogenous non-histidine codon with a histidine codon.

In various aspects, methods of making and using the non-human animalsare also provided. When immunized with an antigen of interest, thegenetically modified non-human animals are capable of generating B cellpopulations that produce antigen-binding proteins comprising heavy chainvariable domains with histidine residues, wherein the antigen-bindingproteins exhibit enhanced pH-dependent recycling and/or increased serumhalf-life. In various embodiments, the non-human animals generate B cellpopulations that express human heavy chain variable domains along withcognate human light chain variable domains. In various embodiments, thegenetically modified immunoglobulin heavy chain loci are present in agermline genome of the non-human animal.

In various embodiments, the genetically modified immunoglobulin heavychain locus comprises a modification that deletes or renders, all orsubstantially all, non-functional endogenous V_(H), D, and J_(H) genesegments; and the genetically modified locus comprises an unrearrangedheavy chain variable region nucleotide sequence comprising one or morehuman V_(H), D, and/or J_(H) gene segments having one or more histidinecodons, wherein the unrearranged heavy chain variable region nucleotidesequence is present at an endogenous location (i.e., where thenucleotide sequence is located in a wild-type non-human animal) orpresent ectopically (e.g., at a locus different from the endogenousimmunoglobulin chain locus in its genome, or within its endogenouslocus, e.g., within an immunoglobulin variable locus, wherein theendogenous locus is placed or moved to a different location in thegenome). In one embodiment, e.g., about 80% or more, about 85% or more,about 90% or more, about 95% or more, about 96% or more, about 97% ormore, about 98% or more, or about 99% or more of all endogenous heavychain V, D, or J gene segments are deleted or rendered non-functional.In one embodiment, e.g., at least 95%, 96%, 97%, 98%, or 99% ofendogenous functional heavy chain V, D, or J gene segments are deletedor rendered non-functional.

In one embodiment, the non-human animal is a mammal. Althoughembodiments directed to introducing histidine codons into anunrearranged human heavy chain variable gene sequence in a mouse areextensively discussed herein, other non-human animals are also providedthat comprise a genetically modified immunoglobulin locus containing anunrearranged human heavy chain variable region nucleotide sequencecomprising an addition of least one histidine codon or a substitution ofat least one endogenous non-histidine codon with a histidine codon. Suchnon-human animals include any of those which can be genetically modifiedto express the histidine-containing heavy chain variable domain asdisclosed herein, including, e.g., mouse, rat, rabbit, pig, bovine(e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog,ferret, primate (e.g., marmoset, rhesus monkey), etc. For example, forthose non-human animals for which suitable genetically modifiable EScells are not readily available, other methods are employed to make anon-human animal comprising the genetic modification. Such methodsinclude, e.g., modifying a non-ES cell genome (e.g., a fibroblast or aninduced pluripotent cell) and employing somatic cell nuclear transfer(SCNT) to transfer the genetically modified genome to a suitable cell,e.g., an enucleated oocyte, and gestating the modified cell (e.g., themodified oocyte) in a non-human animal under suitable conditions to forman embryo. Methods for modifying a non-human animal genome (e.g., a pig,cow, rodent, chicken, etc. genome) include, e.g., employing a zincfinger nuclease (ZFN) or a transcription activator-like effectornuclease (TALEN) to modify a genome to include a nucleotides sequencethat encodes

In one embodiment, the non-human animal is a small mammal, e.g., of thesuperfamily Dipodoidea or Muroidea. In one embodiment, the geneticallymodified animal is a rodent. In one embodiment, the rodent is selectedfrom a mouse, a rat, and a hamster. In one embodiment, the rodent isselected from the superfamily Muroidea. In one embodiment, thegenetically modified animal is from a family selected from Calomyscidae(e.g., mouse-like hamsters), Cricetidae (e.g., hamster, New World ratsand mice, voles), Muridae (true mice and rats, gerbils, spiny mice,crested rats), Nesomyidae (climbing mice, rock mice, with-tailed rats,Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), andSpalacidae (e.g., mole rates, bamboo rats, and zokors). In a specificembodiment, the genetically modified rodent is selected from a truemouse or rat (family Muridae), a gerbil, a spiny mouse, and a crestedrat. In one embodiment, the genetically modified mouse is from a memberof the family Muridae. In one embodiment, the animal is a rodent. In aspecific embodiment, the rodent is selected from a mouse and a rat. Inone embodiment, the non-human animal is a mouse.

In one embodiment, the non-human animal is a rodent that is a mouse of aC57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN,C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6N, C57BL/6NJ, C57BL/10,C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola.

In another embodiment, the mouse is a 129 strain. In one embodiment, the129 strain is selected from the group consisting of 129P1, 129P2, 129P3,129X1, 129S1 (e.g., 12951/SV, 12951/SvIm), 129S2, 129S4, 129S5,12959/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2 (see, e.g.,Festing et al. (1999) Revised nomenclature for strain 129 mice,Mammalian Genome 10:836, see also, Auerbach et al. (2000) Establishmentand Chimera Analysis of 129/SvEv- and C57BL/6-Derived Mouse EmbryonicStem Cell Lines). In one embodiment, the genetically modified mouse is amix of an aforementioned 129 strain and an aforementioned C57BL strain(e.g., a C57BL/6 strain). In another embodiment, the mouse is a mix ofaforementioned 129 strains, or a mix of aforementioned C57BL/6 strains.In one embodiment, the 129 strain of the mix is a 129S6 (129/SvEvTac)strain. In another embodiment, the mouse is a mix of a 129/SvEv- and aC57BL/6-derived strain. In a specific embodiment, the mouse is a mix ofa 129/SvEv- and a C57BL/6-derived strain as described in Auerbach et al.2000 BioTechniques 29:1024-1032. In another embodiment, the mouse is aBALB strain, e.g., BALB/c strain. In another embodiment, the mouse is amix of a BALB strain (e.g., BALB/c strain) and another aforementionedstrain.

In one embodiment, the non-human animal is a rat. In one embodiment, therat is selected from a Wistar rat, an LEA strain, a Sprague Dawleystrain, a Fischer strain, F344, F6, and Dark Agouti. In one embodiment,the rat strain is a mix of two or more of a strain selected from thegroup consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, andDark Agouti.

In one embodiment, the non-human animal is a mouse. In one embodiment,the mouse is a VELOCIMMUNE® humanized mouse.

VELOCIMMUNE® humanized mice (see, e.g., U.S. Pat. Nos. 6,596,541,7,105,348, and US20120322108A1, which are incorporated herein byreference in their entireties), which contain a precise replacement ofmouse immunoglobulin variable regions with human immunoglobulin variableregions at the endogenous mouse loci, display a surprising andremarkable similarity to wild-type mice with respect to B celldevelopment. VELOCIMMUNE® humanized mice display an essentially normal,wild-type response to immunization that differed only in one significantrespect from wild-type mice—the variable regions generated in responseto immunization are fully human.

VELOCIMMUNE® humanized mice contain a precise, large-scale replacementof germline variable region nucleotide sequences of mouse immunoglobulinheavy chain (IgH) and immunoglobulin light chain (e.g., κ light chain,Igκ) with corresponding human immunoglobulin variable region nucleotidesequences, at the endogenous loci (see, e.g., U.S. Pat. Nos. 6,596,541,7,105,348, US 20120322108A1, which are incorporated herein by referencein their entireties). In total, about six megabases of mouse loci arereplaced with about 1.5 megabases of human genomic sequence. Thisprecise replacement results in a mouse with hybrid immunoglobulin locithat make heavy and light chains that have a human variable regions anda mouse constant region. The precise replacement of mouse V_(H)-D-J_(H)and Vκ-Jκ segments leave flanking mouse sequences intact and functionalat the hybrid immunoglobulin loci. The humoral immune system of themouse functions like that of a wild-type mouse. B cell development isunhindered in any significant respect and a rich diversity of humanvariable regions is generated in the mouse upon antigen challenge.

VELOCIMMUNE® humanized mice are possible because immunoglobulin genesegments for heavy and κ light chains rearrange similarly in humans andmice, which is not to say that their loci are the same or even nearlyso—clearly they are not. However, the loci are similar enough thathumanization of the heavy chain variable gene locus can be accomplishedby replacing about three million base pairs of contiguous mouse sequencethat contains all the V_(H), D, and J_(H) gene segments with about onemillion bases of contiguous human genomic sequence covering basicallythe equivalent sequence from a human immunoglobulin locus.

In some embodiments, further replacement of certain mouse constantregion nucleotide sequences with human constant region nucleotidesequences (e.g., replacement of mouse heavy chain C_(H)1 nucleotidesequence with human heavy chain C_(H)1 nucleotide sequence, andreplacement of mouse light chain constant region nucleotide sequencewith human light chain constant region nucleotide sequence) results inmice with hybrid immunoglobulin loci that make antibodies that havehuman variable regions and partly human constant regions, suitable for,e.g., making fully human antibody fragments, e.g., fully human Fab′ s.Mice with hybrid immunoglobulin loci exhibit normal variable genesegment rearrangement, normal somatic hypermutation frequencies, andnormal class switching. These mice exhibit a humoral immune system thatis indistinguishable from wild type mice, and display normal cellpopulations at all stages of B cell development and normal lymphoidorgan structures—even where the mice lack a full repertoire of humanvariable region nucleotide segments. Immunizing these mice results inrobust humoral responses that display a wide diversity of variable genesegment usage.

The precise replacement of the mouse germline variable region nucleotidesequence allows for making mice that have partly human immunoglobulinloci. Because the partly human immunoglobulin loci rearrange,hypermutate, and class switch normally, the partly human immunoglobulinloci generate antibodies in a mouse that comprise human variableregions. Nucleotide sequences that encode the variable regions can beidentified and cloned, then fused (e.g., in an in vitro system) with anysequences of choice, e.g., any immunoglobulin isotype suitable for aparticular use, resulting in an antibody or antigen-binding proteinderived wholly from human sequences.

In various embodiments, at least one histidine codon is present in anunrearranged heavy chain variable region nucleotide sequence thatencodes an N-terminal region, a loop 4 region, a CDR1, a CDR2, a CDR3,or a combination thereof.

In various embodiments, at least one histidine codon is present in anunrearranged heavy chain variable region nucleotide sequence thatencodes a framework region (FR) selected from the group consisting ofFR1, FR2, FR3, and FR4.

In various aspects, the genetically modified immunoglobulin locuscomprises a nucleotide sequence wherein at least one codon has beenreplaced with a histidine codon.

In various aspects, the genetically modified immunoglobulin locuscomprises an unrearranged human heavy chain variable region nucleotidesequence comprising a substitution of at least one endogenousnon-histidine codon with a histidine codon.

In one embodiment, 2 or more, 3 or more, 4 or more, 5 or more, 6 ormore, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 ormore, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 ormore, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 ormore, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 ormore, 31 or more, 32 or more, 33 or more, 34 or more, 35 or more, 36 ormore, 37 or more, 38 or more, 39 or more, 40 or more, 41 or more, 42 ormore, 43 or more, 44 or more, 45 or more, 46 or more, 47 or more, 48 ormore, 49 or more, 50 or more, 51 or more, 52 or more, 53 or more, 54 ormore, 55 or more, 56 or more, 57 or more, 58 or more, 59 or more, 60 ormore, or 61 or more of the endogenous non-histidine codons are replacedwith histidine codons.

Previous studies on reading frame usage of human immunoglobulin D genesegments have shown that, of the three reading frames (i.e., stop,hydrophobic, and hydrophilic), the stop frame is used very infrequently.Apparently, some stop frames are chewed back and result in expression.However, stop reading frames are used at such a low frequency that forthe purposes of engineering histidine codons, it is more efficient notto use the stop reading frame. As between hydrophilic and hydrophobicreading frames, the hydrophilic reading frame appears to be preferred.Thus, in one embodiment, the hydrophilic reading frame of human D genesegments is engineered to contain one or more histidine codons (ascompared with the stop frame or with the hydrophobic frame).

Methods of introducing a mutation in vitro, e.g., site-directedmutagenesis, are well known in the art. In some embodiments of thedescribed invention, histidine codons are enriched by designinghistidine-substituted human D gene segments in silico (e.g., mutation ofY, D, and N codons to H codons, e.g., CAT, CAC), which are synthesized(e.g., chemical synthesis) with (unique) restriction enzyme sites forligating them back together. The synthesized D gene segments are madewith the appropriate recombination signal sequences (RSS) upstream anddownstream. In one embodiment, when ligated to one another, thesynthesized histidine-substituted D gene segments include the intergenicsequences observed in a human between each D gene segment.

It is understood that the codons that encode the one or more histidines,upon rearrangement and/or somatic hypermutation, may change such thatone or more of the histidines will be changed to another amino acid.However, this may not occur for each and every codon encoding histidine,in each and every rearrangement in the non-human animal. If such changesoccur, the changes may occur in some but not all B cells or in some butnot all heavy chain variable sequences.

In various aspects, the genetically modified immunoglobulin locuscomprises a human heavy chain V, D, and J gene segment, wherein at leastone of the human D gene segment has been inverted 5′ to 3′ with respectto a corresponding wild-type sequence, and wherein at least one readingframe of the inverted human D gene segment comprises a histidine codon.

In various embodiments, the nucleotide sequence comprises one or more, 2or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 ormore, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 ormore, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 ormore, 21 or more, 22 or more, 23 or more, 24 or more, or 25 or more ofhistidine codons.

There are 25 functional human D gene segments in 6 families of 3-5members each (one family- the D7 family- has a single member). Directrecombination of human D gene segments is much more frequent thaninversion, although inverted reading frames exhibit more histidinecodons. Certain D gene segments and reading frames are used morefrequently than others. All three direct reading frames and all threeinverted orientation reading frames for all the functional D genesegments are presented in FIGS. 10A-10E. As shown in FIGS. 10A-10E,there are many more histidine codons in inverted reading frames than indirect reading frames. More specifically, there are 34 histidines ininverted reading frames and only four in direct reading frames. Inaddition, of the four in direct reading frames, three histidines areencoded by pseudogenes or present in alternate alleles. Therefore, thereis only a single direct reading frame of a germline human D gene segmentthat contains a histidine codon, with further histidine codons possiblyencountered in alternate alleles (presumably in subsets of the humanpopulation).

Inverted D rearrangements are extremely rare. Tuaillon et al. (J.Immunol., 154(12): 5453-6465, incorporated by reference herein in itsentirety) showed that usage of inverted reading frames (as measured bylimiting dilution PCT) is very rare, i.e., that the ratio of direct toindirect rearrangements are, in most cases, 100 to 1000. To the extentthat the ratio of direct to indirect rearrangement was low, it was onlyobserved in those D segments that exhibit very low usage. It was alsoshown that D gene segment family 7, which is located adjacent to J1 (fardown from other D family members) is mostly used in fetuses, butexhibits a low usage in adults (Schroeder et al., Immunology 30, 2006,119-135, incorporated by reference herein in its entirety). Therefore,in one embodiment, D family 7 sequences are not inverted 5′ to 3′.

In one embodiment, at least two, at least three, at least four, at leastfive, at least six, at least seven, at least eight, at least nine, atleast ten, at least eleven, at least twelve, at least thirteen, at leastfourteen, at least fifteen, at least sixteen, at least seventeen, atleast eighteen, at least nineteen, at least twenty, at least twenty one,at least twenty two, at least twenty three, at least twenty four, or allor substantially all of the human functional D gene segments areinverted 5′ to 3′ with respect to corresponding wild type sequences.

In one embodiment, the human immunoglobulin heavy chain variable domaincomprising at least one non-naturally occurring histidine residueexhibits pH-dependent antigen binding characteristics. For example, anantibody comprising the modified immunoglobulin heavy chain variabledomain binds a target with sufficient affinity at around a neutral pH(e.g., pH of about 7.0 to about 7.4), but either does not bind or bindsweaker to the same target at an acidic pH (e.g., pH of about 5.5 toabout 6.0). In one embodiment, the acidic pH is selected from about 5.5,about 5.6, about 5.7, about 5.8, about 5.9, and about 6.0. In oneembodiment, the neutral pH is selected from about 7.0, about 7.1, about7.2, about 7.3, and about 7.4.

In one embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 2 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 25° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin heavy chain locus as described herein has adissociative half-life (t_(1/2)) of less than 2 min at an acidic pH(e.g., pH of about 5.5 to about 6.0) at 37° C. In one embodiment, anantigen-binding protein comprising a heavy chain variable domainexpressed by the genetically modified immunoglobulin heavy chain locusas described herein has a dissociative half-life (t_(1/2)) of less than1 min at an acidic pH (e.g., pH of about 5.5 to about 6.0) at 25° C. Inone embodiment, an antigen-binding protein comprising a heavy chainvariable domain expressed by the genetically modified immunoglobulinheavy chain locus as described herein has a dissociative half-life(t_(1/2)) of less than 1 min at an acidic pH (e.g., pH of about 5.5 toabout 6.0) at 37° C. In one embodiment, an antigen-binding proteincomprising a heavy chain variable domain expressed by the geneticallymodified immunoglobulin locus as described herein has at least about2-fold, at least about 3-fold, at least about 4-fold, at least about5-fold, at least about 10-fold, at least about 15-fold, at least about20-fold, at least about 25-fold, or at least about 30-fold decrease indissociative half-life (t_(1/2)) at an acidic pH (e.g., pH of about 5.5to about 6.0) as compared to the dissociative half-life (t_(1/2)) of theantigen-binding protein at a neutral pH (e.g., pH of about 7.0 to about7.4).

In one embodiment, antigen binding proteins comprising the geneticallymodified human immunoglobulin heavy chain variable domain is capable ofspecifically binding an antigen of interest with an affinity (K_(D))lower than 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or 10⁻¹⁰, 10⁻¹¹, 10⁻¹² at a neutral orphysiological pH (pH of about 7.0 to about 7.4).

The altered binding property of the immunoglobulin heavy chain variabledomain at an acidic pH (e.g., pH of about 5.5 to about 6.0) would, insome circumstances, allow faster turnover of the antibody because thetherapeutic antibody will bind a target on a cell's surface, beinternalized into an endosome, and more readily or more rapidlydissociate from the target in the endosome, so that the therapeutic canbe recycled to bind yet another molecule of target present in anothercell. This would allow one to administer a therapeutic antibody at alower dose, or administer the therapeutic antibody less frequently. Thisis particularly useful in a situation where it is not desirable toadminister a therapeutic antibody frequently, or administer at a levelabove a certain dosage for safety or toxicity reasons.

In various embodiments, the human immunoglobulin heavy chain variableregion nucleotide sequence as described herein is operably linked to ahuman or non-human heavy chain constant region nucleotide sequence(e.g., a heavy chain constant region nucleotide sequence that encodes animmunoglobulin isotype selected from IgM, IgD, IgG, IgE, and IgA). Invarious embodiments, the human or non-human heavy chain constant regionnucleotide sequence is selected from the group consisting of a C_(H)1, ahinge, a C_(H2), a C_(H3), and a combination thereof. In one embodiment,the constant region nucleotide sequence comprises a C_(H)1, a hinge, aC_(H)2, and a C_(H)3 (e.g., C_(H)1-hinge-a C_(H)2-C_(H)3).

In various embodiments, the heavy chain constant region nucleotidesequence is present at an endogenous locus (i.e., where the nucleotidesequence is located in a wild-type non-human animal) or presentectopically (e.g., at a locus different from the endogenousimmunoglobulin chain locus in its genome, or within its endogenouslocus, e.g., within an immunoglobulin variable locus, wherein theendogenous locus is placed or moved to a different location in thegenome).

In one embodiment, the heavy chain constant region nucleotide sequencecomprises a modification in a C_(H)2 or a C_(H)3, wherein themodification increases the affinity of the heavy chain constant regionamino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

The neonatal Fc receptor for IgG (FcRn) has been well characterized inthe transfer of passive humoral immunity from a mother to her fetusacross the placenta and proximal small intestine (Roopenian, D. andAkilesh, S., Nat. Rev. Immun., 2007, 7:715-725, which is incorporated byreference herein in its entirety). FcRn binds to the Fc portion of IgGat a site that is distinct from the binding sites of the classical FcγRsor the C1q component of complement, which initiates the classicalpathway of complement activation. More specifically, it was shown thatFcRn binds the C_(H)2-C_(H)3 hinge region of IgG antibodies—a versatileregion of Fc that also binds Staphylococcal protein A, Streptococcalprotein G, and the rheumatoid factor. In contrast to other Fc-bindingproteins, however, FcRn binds the Fc region of IgG in a strictlypH-dependent manner; at physiological pH 7.4, FcRn does not bind IgG,whereas at the acidic pH of the endosome (e.g., where the pH ranges fromabout 5.5 to about 6.0), FcRn exhibits a low micromolar to nanomolaraffinity for the Fc region of IgG. This pH-dependent interaction hasbeen shown to be mediated by the titration of histidine residues in theC_(H)2-C_(H)3 region of IgG and their subsequent interaction with acidicresidue on the surface of FcRn (Roopenian, D. and Akilesh, S., Nat. Rev.Immun., 2007, 7:715-725, incorporated by reference in its entirety).

Various mutations in the C_(H)2-C_(H)3 region of IgG that can increasethe affinity of Fc region to FcRn at an acidic pH are known in the art.These include, but are not limited to, modification at position 250(e.g., E or Q); 250 and 428 (e.g., L or F); 252 (e.g., L/Y/F/W or T),254 (e.g., S or T), and 256 (e.g., S/R/Q/E/D or T); or a modification at428 and/or 433 (e.g., L/R/S/P/Q or K) and/or 434 (e.g., H/F or Y); or amodification at 250 and/or 428; or a modification at 307 or 308 (e.g.,308F, V308F), and 434. In another example, the modification can comprisea 428L (e.g., M428L) and 434S (e.g., N434S) modification; a 428L, 2591(e.g., V259I), and a 308F (e.g., V308F) modification; a 433K (e.g.,H433K) and a 434 (e.g., 434Y) modification; a 252, 254, and 256 (e.g.,52Y, 254T, and 256E) modification; a 250Q and 428L modification, or acombination thereof.

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 252 and 257,wherein the modification increases the affinity of the human C_(H)2amino acid sequence to FcRn in an acidic environment (e.g., in anendosome where pH ranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)2 amino acid sequence comprising at least onemodification between amino acid residues at positions 307 and 311,wherein the modification increases the affinity of the C_(H)2 amino acidsequence to FcRn in an acidic environment (e.g., in an endosome where pHranges from about 5.5 to about 6.0).

In one embodiment, the heavy chain constant region nucleotide sequenceencodes a human C_(H)3 amino acid sequence, wherein the C_(H)3 aminoacid sequence comprises at least one modification between amino acidresidues at positions 433 and 436, wherein the modification increasesthe affinity of the C_(H)3 amino acid sequence to FcRn in an acidicenvironment (e.g., in an endosome where pH ranges from about 5.5 toabout 6.0).

In one embodiment, the human constant region amino acid sequence encodedby the heavy chain constant region nucleotide sequence described hereincomprises a mutation selected from the group consisting of M428L, N434S,and a combination thereof. In one embodiment, the human constant regionamino acid sequence comprises a mutation selected from the groupconsisting of M428L, V259I, V308F, and a combination thereof. In oneembodiment, the human constant region amino acid sequence comprises anN434A mutation. In one embodiment, the human constant region amino acidsequence comprises a mutation selected from the group consisting ofM252Y, S254T, T256E, and a combination thereof. In one embodiment, thehuman constant region amino acid sequence comprises a mutation selectedfrom the group consisting of T250Q, M248L, or both. In one embodiment,the human constant region amino acid sequence comprises a mutationselected from the group consisting of H433K, N434Y, or both.

In one embodiment, the heavy chain constant region amino acid sequenceis a non-human constant region amino acid sequence, and the heavy chainconstant region amino acid sequence comprises one or more of any of thetypes of modifications described above.

In one embodiment, the heavy chain constant region nucleotide sequenceis a human heavy chain constant region amino acid sequence, and thehuman heavy chain constant region amino acid sequence comprises one ormore of any of the types of modifications described above.

Engineered Histidine Residues in Immunoglobulin Light Chain Genes

In various embodiments, genetically modified non-human animals (e.g.,mammals, e.g., mice, rats, rabbits, etc.) are provided that comprise intheir genome, e.g., in their germline, nucleotide sequence(s) encodinghuman antibody molecules that exhibit pH-dependent antigen binding,e.g., a nucleotide sequence of immunoglobulin light chain comprisingrearranged human immunoglobulin light chain variable region sequenceencoding antibodies that exhibits pH-dependent antigen binding; embryos,cells, and tissues comprising the same; methods of making the same; aswell as methods of using the same.

The inventors have discovered that non-human animals that expressantibodies that are capable of binding to an antigen in a pH dependentmanner can be made by making modifications of an immunoglobulin lightchain variable region at one or more positions along the sequence of thelight chain. Methods of making modifications in the germline of anon-human animal so that the animal would express histidines in CDRs ofantibodies are described. In particular, methods for makingmodifications in an immunoglobulin light chain variable sequence in thegermline of the mouse are described. Variable region sequence, e.g., oflight chains, typically show somatic hypermutation along the variableregion sequence, and, in some cases, such mutations can result in asubstitution of histidine residues (see, e.g., FIG. 15). Such mutationscan even occur in complementary determining regions (CDRs), which arethe regions of variable domains responsible for antigen binding. In somecases, such mutations can result in antibodies that display pH-dependentantigen binding, e.g., reduced antigen binding at an acidic pH ascompared to antigen binding at a neutral pH. Such pH-dependent antigenbinding is desired because it may enable the antibody to bind to theantigen outside the cell, and, when internalized into an endosome,release the antigen and recycle back to the surface to bind anotherantigen, avoiding target-mediated clearance. Approaches for introducinghistidine residues to achieve this effect by using a random his-scanningmutagenesis to engineer pH-dependent binding properties in anti-IL-6Rantibodies have been reported (US 2011/0111406 A1). However, randommutagenesis of antibody residues may result in decreased affinity ofantibody to the antigen. A non-human animal genetically modified toexpress a histidine substitution in antibody sequence enables generationof high-affinity antibodies in response to an antigen of interest that,due to histidine modification(s), would also display pH-dependentantigen binding.

Thus, in various embodiments, provided herein is a genetically modifiednon-human animal (e.g., rodent, e.g., a mouse or a rat) that comprisesin its genome, e.g., its germline, a human immunoglobulin light chainvariable region sequence comprising modifications that result in theanimal expressing antibodies capable of binding to antigens in apH-dependent manner. In one embodiment, the non-human animal comprisesmodifications in the human immunoglobulin light chain variable regionsequence (e.g., V_(L) and/or J_(L) segment sequence) that comprisesubstitutions in at least one non-histidine codon with a histidine codon(in some cases, also may be referred to as “histidine substitution,”“histidine codon substitution,” or the like). In one embodiment, theanimal comprises at least one substitution of a non-histidine codon witha histidine codon in a nucleotide sequence of a complementarydetermining region (CDR; e.g., CDR1, CDR2, and/or CDR3) of a humanimmunoglobulin light chain. In one embodiment, the substitution is in aCDR3 codon. In one embodiment, the light chain is a κ light chain. Inone embodiment, the animal expresses an immunoglobulin light chain,e.g., a light chain CDR, e.g., a light chain CDR3, comprising asubstitution of at least one amino acid with a histidine. In anotherembodiment, the light chain is a λ light chain. In yet anotherembodiment, the mouse comprises a substitution of at least onenon-histidine codon with a histidine codon in both κ and λ light chains.

A histidine residue is encoded by two different codons, CAT and CAC(deoxyribonucleic acid residues). Thus, a non-histidine codon may besubstituted with a CAT or a CAC. The substitution is engineered in acodon that in its germline configuration (i.e., non-somatically mutatedstate) does not encode a histidine residue.

In one embodiment a light chain is a universal light chain (also termeda common light chain). As described in U.S. patent application Ser. Nos.13/022,759, 13/093,156, 13/412,936 and 13/488,628 (U.S. ApplicationPublication Nos. 2011/0195454, 2012/0021409, 2012/0192300 and2013/0045492, all incorporated herein by reference), a non-human animal(e.g., a mouse) that selects a common light chain for a plurality ofheavy chains has a practical utility. In various embodiments, antibodiesexpressed in a non-human animal comprising only a common light chainwill have heavy chains that can associate and express with an identicalor substantially identical light chain. This is particularly useful inmaking bispecific antibodies. For example, such an animal can beimmunized with a first immunogen to generate a B cell that expresses anantibody that specifically binds a first epitope. The animal (or ananimal genetically the same) can be immunized with a second immunogen togenerate a B cell that expresses an antibody that specifically binds thesecond epitope. Variable heavy chain regions can be cloned from the Bcells and expressed with the same heavy chain constant region and thesame light chain (e.g., a common light chain) in a cell to make abispecific antibody, wherein the heavy chain component of the bispecificantibody has been selected by an animal to associate and express withthe same light chain component. In various embodiments described, thevariable regions of the genetically engineered mice are human variableregions.

Thus, a mouse was engineered that is capable of generatingimmunoglobulin light chains that will suitably pair with a ratherdiverse family of heavy chains, including heavy chains whose humanvariable regions depart from germline sequences, e.g., affinity maturedor somatically mutated variable regions. In various embodiments, themouse is devised to pair human light chain variable domains with humanheavy chain variable domains that comprise somatic mutations, thusenabling a route to high affinity binding proteins suitable for use ashuman therapeutics.

The genetically engineered mouse, through the long and complex processof antibody selection within an organism, makes biologically appropriatechoices in pairing a diverse collection of human heavy chain variabledomains with a limited number of human light chain options. In order toachieve this, the mouse is engineered to present a limited number ofhuman light chain variable domain options in conjunction with a widediversity of human heavy chain variable domain options. Upon challengewith an immunogen, the mouse maximizes the number of solutions in itsrepertoire to develop an antibody to the immunogen, limited largely orsolely by the number or light chain options in its repertoire. Invarious embodiments, this includes allowing the mouse to achievesuitable and compatible somatic mutations of the light chain variabledomain that will nonetheless be compatible with a relatively largevariety of human heavy chain variable domains, including in particularsomatically mutated human heavy chain variable domains.

The engineered common light chain mouse described in U.S. ApplicationPublication Nos. 2011/0195454, 2012/0021409, 2012/0192300 and2013/0045492 comprised nucleic acid sequence encoding a limitedrepertoire of light chain options, e.g., common or universal light chain“ULC” that comprised no more than two V_(L) segments or a singlerearranged human immunoglobulin light chain variable region sequence. Toachieve such limited repertoire, a mouse was engineered to rendernonfunctional or substantially nonfunctional its ability to make, orrearrange, a native mouse light chain variable domain. In one aspect,this was achieved, e.g., by deleting the mouse's light chain variableregion gene segments. As previously described, the endogenous mouselocus can then be modified by exogenous suitable human light chainvariable region gene segments of choice, operably linked to theendogenous mouse light chain constant domain, in a manner such that theexogenous human variable region gene segments can combine with theendogenous mouse light chain constant region gene and form a rearrangedreverse chimeric light chain gene (human variable, mouse constant). Invarious embodiments, the light chain variable region is capable of beingsomatically mutated. In various embodiments, to maximize ability of thelight chain variable region to acquire somatic mutations, theappropriate enhancer(s) is retained in the mouse. In one aspect, inmodifying a mouse κ light chain locus to replace endogenous mouse κlight chain gene segments with human κ light chain gene segments, themouse κ intronic enhancer and mouse κ 3′ enhancer are functionallymaintained, or undisrupted.

Thus, provided was a genetically engineered mouse that expresses alimited repertoire of reverse chimeric (human variable, mouse constant)light chains associated with a diversity of reverse chimeric (humanvariable, mouse constant) heavy chains. In various embodiments, theendogenous mouse κ light chain gene segments are deleted and replacedwith a single (or two) rearranged human light chain region, operablylinked to the endogenous mouse Cκ gene. In embodiments for maximizingsomatic hypermutation of the rearranged human light chain region, themouse κ intronic enhancer and the mouse κ 3′ enhancer are maintained. Invarious embodiments, the mouse also comprises a nonfunctional λ lightchain locus, or a deletion thereof or a deletion that renders the locusunable to make a λ light chain.

The universal light chain mouse generated antibodies in response tovarious antigens that were capable of utilizing a diverse repertoire ofheavy chain variable region sequences, comprising a diverse repertoireof V_(H), D_(H), and J_(H) segments. Antibodies generated in suchgenetically engineered ULC mouse are useful for designing bispecifictherapeutic antibodies; however, as with any other antibody, eachbispecific antibody may only bind to one target during its lifetime inthe plasma; the antibody is internalized into an endosome and targetedfor lysosomal degradation. Studies have shown that MHC-class-1-like Fcγreceptor FcRn is capable of rescuing immunoglobulins from lysosomaldegradation by recycling it back to the cell surface from the sortingendosome. Simister and Mostov (1989) An Fc receptor structurally relatedto MHC class I antigens. Nature 337: 184-87. As explained above, toimprove efficiency of antibody recycling, further modifications toantibody sequences, e.g., modifications that result in decreased antigenbinding at acidic pH (e.g., pH of the endosome), while retainingantibody-antigen affinity and specificity at neutral pH (e.g., pH ofbody fluids such as blood) are beneficial. The non-human animalsdescribed herein, wherein histidine residues are substituted fornon-histidine residues in the a universal light chain sequence arebeneficial because they are capable of producing high-affinityantibodies based on universal light chain format that also displaypH-dependent binding, e.g., display reduced binding to the antigen atacidic versus neutral pH.

Thus, in one embodiment, provided herein is a non-human animal (e.g., arodent, e.g., a mouse or a rat) that comprises in its genome, e.g., inits germline, a limited repertoire of human light chain variableregions, or a single human light chain variable region, from a limitedrepertoire of human light chain variable gene segments, wherein thehuman light chain variable region(s) comprise at least one substitutionof a non-histidine codon for a histidine codon. In some embodiments,provided non-human animals are genetically engineered to include asingle unrearranged human light chain variable region gene segment (ortwo human light chain variable region gene segments) that rearranges toform a rearranged human light chain variable region gene (or tworearranged light chain variable region genes) that expresses a singlelight chain (or that express either or both of two light chains),wherein the light chain variable region gene(s) comprise a substitutionof at least one non-histidine codon with a histidine codon. Therearranged human light chain variable domains encoded by thesehistidine-substituted light chain variable region gene(s) are capable ofpairing with a plurality of affinity-matured human heavy chains selectedby the animals, wherein the heavy chain variable regions specificallybind different epitopes. In various embodiments, the at least onesubstitution of a non-histidine residue with a histidine residue resultsin a rearranged human light chain that, when expressed with a cognateheavy chain, binds to its antigen in a pH-dependent manner.

Genetically engineered animals are provided that express a limitedrepertoire of human light chain variable domains, or a single humanlight chain variable domain, from a limited repertoire of human lightchain variable region gene sequences, wherein the variable region genesequences comprise at least one substitution of a non-histidine codonwith a histidine codon. In some embodiments, provided animals aregenetically engineered to include a single V/J human light chainsequence (or two V/J sequences) that comprises a substitution of atleast one non-histidine codon with a histidine codon and expresses avariable region of a single light chain (or that express either or bothof two variable regions). In one aspect, a light chain comprising thevariable sequence is capable of pairing with a plurality ofaffinity-matured human heavy chains clonally selected by the animal,wherein the heavy chain variable regions specifically bind differentepitopes. In one embodiment, the antibody binds to its antigen(s) in apH-dependent manner. In one embodiment, the single V/J human light chainsequence is selected from Vκ1-39Jκ5 and Vκ3-20Jκ1. In one embodiment,the two V/J sequences are Vκ1-39Jκ5 and Vκ3-20Jκ1. In one embodiment,the Vκ1-39Jκ5 and Vκ3-20Jκ1 sequences are rearranged V/J sequences.

In one aspect, a genetically modified non-human animal is provided thatcomprises a single human immunoglobulin light chain V_(L) gene segmentthat is capable of rearranging with a human J_(L) gene segment (selectedfrom one or a plurality of J_(L) segments) and encoding a human variabledomain of an immunoglobulin light chain, wherein the single humanimmunoglobulin light chain V_(L) gene segment and/or human J_(L) genesegment comprise a substitution of at least one non-histidine codon witha histidine codon. In another aspect, a genetically modified mouse isprovided that comprises no more than two human V_(L) gene segments, eachof which is capable of rearranging with a human J_(L) gene segment(selected from one or a plurality of J_(L) segments) and encoding ahuman variable domain of an immunoglobulin light chain, wherein each ofthe no more than two V_(L) gene segments and/or the J_(L) gene segmentcomprise a substitution of at least one non-histidine residue with ahistidine residue.

Also provided herein is a genetically modified non-human animal thatcomprises in its genome, e.g., in its germline, a single rearrangedhuman immunoglobulin light chain variable region sequence comprising ahuman V_(L) and J_(L) sequences wherein the single rearranged humanimmunoglobulin light chain variable region comprises a substitution ofat least one non-histidine codon with a histidine codon. In one aspect,the single rearranged human immunoglobulin light chain variable regionsequence is derived from human germline V_(L) and J_(L) gene sequences,but for the histidine substitution(s). In one embodiment, the humanimmunoglobulin light chain is a human immunoglobulin κ chain. Thus, inone embodiment, the human V_(L) gene sequence is selected from Vκ1-39and Vκ3-20. In one embodiment, the single rearranged humanimmunoglobulin light chain variable region sequence comprises rearrangedVκ1-39/J or Vκ3-20/J sequence. In one embodiment, the human J_(L) genesequence is selected from Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5. In one embodimentthe human J_(L) sequence is selected from Jκ1 and Jκ5. In oneembodiment, the single rearranged human immunoglobulin light chainvariable region sequence is selected from Vκ1-39R5 and Vκ3-20Jκ1 (e.g.,but for the histidine substitution(s)). In an alternative embodiment,the human immunoglobulin light chain is a human λ chain.

In one embodiment, the substitution of at least one non-histidine codonfor a histidine codon is in the nucleotide sequence encoding acomplementary determining region (CDR) of the light chain variabledomain. In one embodiment, the substitution of at least onenon-histidine codon for a histidine codon is in the nucleotide sequenceencoding CDR1, CDR2 or CDR3 of the light chain variable domain. In onespecific embodiment, the substitution is in the nucleotide sequenceencoding CDR3.

In one aspect, the substitution is of at least one non-histidine codonfor a histidine codon in the CDR3 codon of the human light chainvariable region gene sequence. In one embodiment, the substitution is ofone, two, three, four, or more CDR3 codons. In the embodiment whereinthe single rearranged human immunoglobulin light chain variable regionis a Vκ1-39R5 variable region, the replacement of at least onenon-histidine codon with a histidine codon comprises a replacement at aposition in the immunoglobulin light chain gene sequence encoding CDR3designed to express histidine at position selected from 105, 106, 108,111, and a combination thereof. In one embodiment, the replacement isdesigned to express histidine at positions 105 and 106. In oneembodiment, the replacement is designed to express histidine atpositions 105 and 111. In one embodiment, the replacement is designed toexpress histidine at positions 105 and 108. In one embodiment, thereplacement is designed to express histidine at positions 105, 108 and111. In one embodiment, the replacement is designed to express histidineat positions 105, 106, and 108. In one embodiment, the replacement isdesigned to express histidine at positions 106 and 108. In oneembodiment, the replacement is designed to express histidine atpositions 106 and 111. In one embodiment, the replacement is designed toexpress histidine at positions 108 and 111. In one embodiment, thereplacement is designed to express histidine at positions 106, 108, and111. In yet another embodiment, the replacement is designed to expresshistidine at positions 106, 108 and 111. In one embodiment, thereplacement is designed to express histidine at positions 105, 106, and111. In one embodiment, the replacement is designed to express histidineat positions 105, 106, 108, and 111. The nucleic acid and amino acidsequences of the histidine-substituted regions are depicted in sequencealignment of FIG. 16 and set forth in SEQ ID NOs: 327-357.

In the embodiment wherein the single rearranged human immunoglobulinlight chain variable region is a Vκ3-20Jκ1 variable region, thereplacement of at least one non-histidine codon with a histidine codoncomprises a replacement at a position in the immunoglobulin light chaingene sequence encoding CDR3 region that is designed to express histidineat position selected from 105, 106, 107, 109, and a combination thereof.In one embodiment, the replacement is designed to express histidine atpositions 105 and 106. In one embodiment, the replacement is designed toexpress histidine at positions 105 and 107. In one embodiment, thereplacement is designed to express histidine at positions 105 and 109.In one embodiment, the replacement is designed to express histidine atpositions 106 and 107. In one embodiment, the replacement is designed toexpress histidine at positions 106 and 109. In one embodiment, thereplacement is designed to express histidine at positions 107 and 109.In one embodiment, the replacement is designed to express histidine atpositions 105, 106, and 107. In one embodiment, the replacement isdesigned to express histidine at positions 105, 107, and 109. In oneembodiment, the replacement is designed to express histidine atpositions 106, 108, and 111. In one embodiment, the replacement isdesigned to express histidine at positions 105, 106 and 109. In anotherembodiment, the replacement is designed to express histidine atpositions 105, 106, 107, and 109. The nucleic acid and amino acidsequences of exemplary histidine-substituted regions are depicted insequence alignment of FIG. 27 and set forth in SEQ ID NOs: 398-403.

Amino acid positions (105, 106, etc.) are based on a unique numberingdescribed in Lefranc et al. (2003) Dev. Comp. Immunol. 27:55-77, and canalso be viewed on www.imgt.org.

In one embodiment, the human V_(L) gene segment is operably linked to ahuman or non-human leader sequence. In one embodiment, the leadersequence is a non-human leader sequence. In a specific embodiment, thenon-human leader sequence is a mouse Vκ3-7 leader sequence. In aspecific embodiment, the leader sequence is operably linked to anunrearranged human V_(L) gene segment. In a specific embodiment, theleader sequence is operably linked to a rearranged human V_(L)/J_(L)sequence. Thus, in one specific embodiment, the single rearrangedVκ1-39/Jκ5 or Vκ3-20/Jκ1 variable region gene sequence comprising atleast one histidine substitution is operably linked to a mouse Vκ3-7leader sequence.

In one embodiment, the V_(L) gene segment is operably linked to animmunoglobulin promoter sequence. In one embodiment, the promotersequence is a human promoter sequence. In a specific embodiment, thehuman immunoglobulin promoter is a human Vκ3-15 promoter. In a specificembodiment, the promoter is operably linked to an unrearranged humanV_(L) gene segment. In a specific embodiment, the promoter is operablylinked to a rearranged human V_(L)/J_(L) sequence. Thus, in one specificembodiment, the single rearranged Vκ1-39/Jκ5 or Vκ3-20/Jκ1 variableregion gene sequence comprising at least one histidine substitution isoperably linked to the human Vκ3-15 promoter.

In one embodiment, the light chain locus comprises a leader sequenceflanked 5′ (with respect to transcriptional direction of a V_(L) genesegment) with a human immunoglobulin promoter and flanked 3′ with ahuman V_(L) gene segment that rearranges with a human J_(L) segment andencodes a variable domain of a reverse chimeric light chain comprisingan endogenous non-human light chain constant region (C_(L)). In aspecific embodiment, the V_(L) and J_(L) gene segments are at thenon-human Vκ locus, and the non-human C_(L) is a non-human Cκ (e.g.,mouse Cκ). In one specific embodiment, the variable region sequence isoperably linked to the non-human constant region sequence, e.g., thenon-human Cκ gene sequence.

In one embodiment, the light chain locus comprises a leader sequenceflanked 5′ (with respect to transcriptional direction of a V_(L) genesegment) with a human immunoglobulin promoter and flanked 3′ with arearranged human variable region sequence (V_(L)/J_(L) sequence) andencodes a variable domain of a reverse chimeric light chain comprisingan endogenous non-human light chain constant region (C_(L)). In aspecific embodiment, the rearranged human V_(L)/J_(L) sequence is at thenon-human kappa (κ) locus, and the non-human C_(L) is a non-human Cκ. Inone specific embodiment, the rearranged human variable region sequenceis operably linked to the non-human immunoglobulin light chain constantregion sequence, e.g., the non-human Cκ gene sequence. In oneembodiment, the non-human immunoglobulin light chain constant regionsequence is an endogenous non-human sequence. In one embodiment, thenon-human animal is a mouse and the Cκ gene sequence is a mouse Cκ genesequence. In one embodiment, the rearranged human immunoglobulin lightchain variable region sequence comprising a substitution of at least onenon-histidine codon with a histidine codon is at the endogenousnon-human (e.g., mouse) immunoglobulin light chain locus (κ locus).Exemplary embodiments of the locus are presented in FIGS. 23C, 23E, 29C,and 29D.

In one embodiment, the genetically modified non-human animal is a mouse,and the variable region locus of the mouse is a κ light chain locus, andthe κ light chain locus comprises a mouse κ intronic enhancer, a mouse κ3′ enhancer, or both an intronic enhancer and a 3′ enhancer.

In one embodiment, the non-human animal (e.g., a rodent, e.g., a rat ora mouse) comprises a nonfunctional immunoglobulin lambda (2) light chainlocus. In a specific embodiment, the λ light chain locus comprises adeletion of one or more sequences of the locus, wherein the one or moredeletions renders the λ light chain locus incapable of rearranging toform a light chain gene. In another embodiment, all or substantially allof the V_(L) gene segments of the λ light chain locus are deleted. Inone embodiment, the non-human animal (e.g., rodent, e.g. mouse or rat)comprises a rearranged human immunoglobulin light chain variable regionsequence comprising a substitution of at least one non-histidine codonwith a histidine codon, and lacks a functional unrearrangedimmunoglobulin light chain variable region, e.g., endogenousunrearranged light chain variable region. In one embodiment, therearranged, histidine-substituted human immunoglobulin light chainvariable region gene sequence replaces endogenous unrearrangedimmunoglobulin light chain variable region gene sequence.

In one embodiment, the animal makes a light chain that comprises asomatically mutated variable domain derived from a human variable regionsequence that comprises a substitution of at least one non-histidinecodon with a histidine codon. In one embodiment, the light chaincomprises a somatically mutated variable domain derived from a humanvariable region sequence that comprises a substitution of at least onenon-histidine codon with a histidine codon, and a non-human Cκ region.In one embodiment, the non-human animal does not express a λ lightchain.

One skilled in the art would appreciate that although substitution(s) ofat least one non-histidine residue with a histidine residue isgenetically engineered into the human immunoglobulin light chainvariable region, due to somatic hypermutations, not all antibodies thatare generated in the genetically modified non-human animal will harborthat histidine residue(s) at engineered position(s). However, generationof a wide repertoire of antibodies in the non-human animal will allow toselect for in vivo generated antigen-specific antibodies that displayhigh affinity for an antigen of interest while retaining histidinemodifications introduced into the germline and, thus, exhibitingpH-dependent antigen binding.

Thus, in one embodiment, the animal retains at least one substitution ofa non-histidine amino acid with a histidine. In one embodiment, theanimal retains all or substantially all histidine substitutions in itssomatically mutated light chain variable domain that were introducedinto its variable region gene.

In one embodiment, the genetically modified non-human animal describedherein also comprises in its genome, e.g., its germline, an unrearrangedimmunoglobulin heavy chain variable region comprising V_(H), D_(H), andJ_(H) gene segment sequences. In one embodiment, the V_(H), D_(H), andJ_(H) gene segment sequences are human V_(H), D_(H), and J_(H) genesegment sequences, and the unrearranged immunoglobulin heavy chainvariable region is a human heavy chain variable region. In oneembodiment, the human V_(H), D_(H), and J_(H) gene segment sequences areoperably linked to non-human heavy chain constant region sequence. Inone embodiment, the non-human heavy chain constant region sequence is anendogenous non-human heavy chain constant region sequence. In oneembodiment, the human heavy chain gene segment sequences are at theendogenous non-human immunoglobulin heavy chain locus. In oneembodiment, the human immunoglobulin heavy chain variable regionsequence comprised in a non-human animal also comprises a substitutionof at least one non-histidine codon for a histidine codon.

In one embodiment, the non-human animal described herein expresses animmunoglobulin light chain that comprises a non-human light chainconstant region sequence.

In one embodiment, the non-human animal expresses an immunoglobulinlight chain that comprises a human light chain constant region sequence.

In one embodiment, the non-human animal described herein expresses animmunoglobulin heavy chain that comprises a non-human sequence selectedfrom a C_(H)1 sequence, a hinge sequence, a C_(H)2 sequence, a C_(H)3sequence, and a combination thereof.

In one embodiment, the non-human animal expresses an immunoglobulinheavy chain that comprises a human sequence selected from a C_(H)1sequence, a hinge sequence, a C_(H)2 sequence, a C_(H)3 sequence, and acombination thereof.

In the embodiment where the animal comprises a single rearrangedimmunoglobulin light chain variable region comprising a substitution ofat least one non-histidine codon with a histidine codon, the rearrangedimmunoglobulin light chain sequence in the germline of the animal is atan endogenous non-human immunoglobulin light chain locus. In a specificembodiment, the rearranged immunoglobulin light chain sequencecomprising a substitution of at least one non-histidine codon with ahistidine codon in the germline of the animal replaces all orsubstantially all endogenous non-human light chain V and J segmentsequences at the endogenous non-human immunoglobulin light chain locus.

In one embodiment, the non-human animal comprises a replacement ofendogenous V_(H) gene segments with one or more human V_(H) genesegments, wherein the human V_(H) gene segments are operably linked to anon-human C_(H) region gene, such that the non-human animal rearrangesthe human V_(H) gene segments and expresses a reverse chimericimmunoglobulin heavy chain that comprises a human V_(H) domain and anon-human C_(H). In one embodiment, 90-100% of unrearranged non-humanV_(H) gene segments are replaced with at least one unrearranged humanV_(H) gene segment. In a specific embodiment, all or substantially all(e.g., 90-100%) of the endogenous non-human V_(H) gene segments arereplaced with at least one unrearranged human V_(H) gene segment. In oneembodiment, the replacement is with at least 19, at least 39, or atleast 80 or 81 unrearranged human V_(H) gene segments. In oneembodiment, the replacement is with at least 12 functional unrearrangedhuman V_(H) gene segments, at least 25 functional unrearranged humanV_(H) gene segments, or at least 43 functional unrearranged human V_(H)gene segments. In one embodiment, the non-human animal comprises areplacement of all non-human D_(H) and J_(H) segments with at least oneunrearranged human D_(H) segment and at least one unrearranged humanJ_(H) segment. In one embodiment, the non-human animal comprises areplacement of all non-human D_(H) and J_(H) segments with allunrearranged human D_(H) segments and all unrearranged human J_(H)segments.

A non-human animal, e.g., a mouse, comprising in its genome, e.g., itsgermline, a limited repertoire of human immunoglobulin light chainvariable regions, e.g., a single rearranged human immunoglobulin lightchain variable region (e.g., Vκ1-39/Jκ5 or Vκ3-20/Jκ1), with asubstitution of at least one non-histidine codon with a histidine codonand a diverse repertoire of unrearranged human V_(H), D_(H), and J_(H)segments is capable of generating antigen binding proteins encoded byheavy chain variable region sequences derived from various permutationsof unrearranged human V_(H), D_(H), and J_(H) segments, wherein theV_(H), D_(H), and J_(H) segments present in the heavy chain variablesequences are derived from all or substantially all functional humanV_(H), D_(H), and J_(H) segments present in the genome of the animal.Various available possibilities for heavy chain variable domainsequences expressed in the cells, e.g., B cells, of the geneticallymodified animals described herein (i.e., derived from combinations ofvarious functional human V, D, and J segments) are described in U.S.Application Publication Nos. 2011/0195454, 2012/0021409, 2012/0192300and 2013/0045492, all incorporated herein by reference. In variousembodiments, the rearranged human immunoglobulin light chain variableregion sequence comprising substitution(s) of at least one non-histidinecodon with a histidine codon and the unrearranged human immunoglobulinheavy chain variable region sequence are comprised in the germline ofthe non-human animal.

In one embodiment, the non-human animal comprises one copy of one orboth of the rearranged human immunoglobulin light chain variable regionsequence comprising substitution(s) of at least one non-histidine codonwith a histidine codon and the unrearranged human immunoglobulin heavychain variable region sequence. In another embodiment, the non-humananimal comprises two copies of one or both of the rearranged humanimmunoglobulin light chain variable region sequence comprisingsubstitution(s) of at least one non-histidine codon with a histidinecodon and the unrearranged human immunoglobulin heavy chain variableregion sequence. Thus, the non-human animal may be homozygous orheterozygous for one or both the rearranged human immunoglobulin lightchain variable region sequence comprising substitution(s) of at leastone non-histidine codon with a histidine codon and the unrearrangedhuman immunoglobulin heavy chain variable region sequence.

In addition to genetically modified non-human animals comprising intheir genome an immunoglobulin light chain variable region gene sequence(e.g., a single rearranged immunoglobulin light chain variable regiongene sequence) comprising substitution of at least one non-histidinecodon with a histidine codon (e.g., in CDR3 of the light chain), alsoprovided herein are genetically modified non-human animals comprising animmunoglobulin light chain variable region gene sequence with one ormore additions of histidine codon(s), such that the expressed variabledomain comprises an additional amino acid(s) which, if not subject tosomatic hypermutation, is a histidine.

The genetically modified non-human animal comprising a humanimmunoglobulin light chain variable region gene sequence with asubstitution of at least one non-histidine codon with a histidine codondescribed herein may be selected from a group consisting of a mouse,rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat,chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey). Forthe non-human animals where suitable genetically modifiable ES cells arenot readily available, methods distinct from those described herein areemployed to make a non-human animal comprising the genetic modification.Such methods include, e.g., modifying a non-ES cell genome (e.g., afibroblast or an induced pluripotent cell) and employing nucleartransfer to transfer the modified genome to a suitable cell, e.g., anoocyte, and gestating the modified cell (e.g., the modified oocyte) in anon-human animal under suitable conditions to form an embryo.

In one aspect, the non-human animal is a mammal. In one aspect, thenon-human animal is a small mammal, e.g., of the superfamily Dipodoideaor Muroidea. In one embodiment, the genetically modified animal is arodent. In one embodiment, the rodent is selected from a mouse, a rat,and a hamster. In one embodiment, the rodent is selected from thesuperfamily Muroidea. In one embodiment, the genetically modified animalis from a family selected from Calomyscidae (e.g., mouse-like hamsters),Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae(true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae(climbing mice, rock mice, with-tailed rats, Malagasy rats and mice),Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., molerates, bamboo rats, and zokors). In a specific embodiment, thegenetically modified rodent is selected from a true mouse or rat (familyMuridae), a gerbil, a spiny mouse, and a crested rat. In one embodiment,the genetically modified mouse is from a member of the family Muridae.In one embodiment, the animal is a rodent. In a specific embodiment, therodent is selected from a mouse and a rat. In one embodiment, thenon-human animal is a mouse.

In a specific embodiment, the non-human animal is a rodent that is amouse of a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa,C57BL/KaLwN, C57BL/6, C57BL/6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10,C57BL/10ScSn, C57BL/10Cr, and C57BL/Ola. In another embodiment, themouse is a 129 strain selected from the group consisting of a strainthat is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm),129S2, 129S4, 129S5, 129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8,129T1, 129T2 (see, e.g., Festing et al. (1999) Revised nomenclature forstrain 129 mice, Mammalian Genome 10:836, see also, Auerbach et al(2000) Establishment and Chimera Analysis of 129/SvEy- andC57BL/6-Derived Mouse Embryonic Stem Cell Lines). In a specificembodiment, the genetically modified mouse is a mix of an aforementioned129 strain and an aforementioned C57BL/6 strain. In another specificembodiment, the mouse is a mix of aforementioned 129 strains, or a mixof aforementioned BL/6 strains. In a specific embodiment, the 129 strainof the mix is a 129S6 (129/SvEvTac) strain. In another embodiment, themouse is a BALB strain, e.g., BALB/c strain. In yet another embodiment,the mouse is a mix of a BALB strain and another aforementioned strain.

In one embodiment, the non-human animal is a rat. In one embodiment, therat is selected from a Wistar rat, an LEA strain, a Sprague Dawleystrain, a Fischer strain, F344, F6, and Dark Agouti. In one embodiment,the rat strain is a mix of two or more strains selected from the groupconsisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and DarkAgouti.

Thus, in one embodiment, the genetically modified non-human animal is arodent. In one embodiment, the genetically modified non-human animal isa rat or a mouse. In one embodiment, the animal is a mouse. Thus, in oneembodiment, provided herein is a genetically modified mouse comprisingin its genome, e.g., its germline, a single rearranged humanimmunoglobulin light chain variable region comprising human V_(L) andJ_(L) gene sequences, wherein the single rearranged human immunoglobulinlight chain variable region comprises a substitution of at leastnon-histidine codon with a histidine codon. In one embodiment, the mouselacks a functional unrearranged immunoglobulin light chain variableregion (e.g., lacks functional unrearranged V and J gene segmentsequences). In one embodiment, the rearranged human immunoglobulin lightchain variable region with histidine codon substitution(s) is Vκ1-39/Jκor Vκ3-20/Jκ variable region. In one embodiment the J segment sequenceis selected from Jκ1, Jκ2, Jκ3, Jκ4, and Jκ5. In one embodiment the Jsegment sequence is Jκ1 or Jκ5. In one embodiment, the substitution ofat least one non-histidine codon with a histidine codon is in thenucleotide sequence encoding a CDR3 region. In one embodiment, whereinthe rearranged variable region sequence is Vκ1-39/Jκ5 sequence, thehistidine substitution(s) is designed to express at a position selectedfrom 105, 106, 108, 111, and a combination thereof. In anotherembodiment, wherein the rearranged variable region sequence isVκ3-20/Jκ1 sequence, the histidine substitution(s) is designed toexpress at a position selected from 105, 106, 107, 109, and acombination thereof. In one embodiment, the rearranged immunoglobulinlight chain variable region with substituted histidine codon(s) isoperably linked to an endogenous mouse immunoglobulin constant regiongene sequence (e.g., Cκ gene sequence). In one embodiment, the mousefurther comprises in its genome, e.g., its germline, an unrearrangedimmunoglobulin heavy chain variable region comprising human V_(H),D_(H), and J_(H) segments. In one embodiment, human V_(H), D_(H), andJ_(H) segments are operably linked to an endogenous mouse immunoglobulinheavy chain constant region gene sequence. In various embodiments, therearranged human immunoglobulin light chain variable region sequencecomprising substitution(s) of at least one non-histidine codon with ahistidine codon and the unrearranged human immunoglobulin heavy chainvariable region sequence are comprised in the germline of the mouse.

Also provided herein are targeting vectors for generating geneticallymodified non-human animals, e.g., mice, described herein. In one aspect,provided is a targeting vector comprising, from 5′ to 3′ intranscriptional direction with reference to the sequences of the 5′ and3′ mouse homology arms of the vector, a 5′ mouse homology arm, a humanor mouse immunoglobulin promoter, a human or mouse leader sequence, ahuman variable region selected from a rearranged human Vκ1-39Jκ5 or arearranged human Vκ3-20Jκ1 and comprising a substitution of at least onenon-histidine codon with a histidine codon, and a 3′ mouse homology arm.In one embodiment, the 5′ and 3′ homology arms target the vector to asequence 5′ with respect to an enhancer sequence that is present 5′ andproximal to the mouse Cκ gene. In another embodiment, the targetingvector comprises a 5′ mouse homology arm followed by a selectioncassette flanked by recombination sites, human or mouse immunoglobulinpromoter, human or mouse leader sequence, a human variable regionselected from a rearranged human Vκ1-39Jκ5 or a rearranged humanVκ3-20Jκ1 and comprising a substitution of at least one non-histidinecodon with a histidine codon, followed by the 3′ mouse homology arm thatcomprises mouse enhancers and constant region (Cκ) sequences.

A selection cassette is a nucleotide sequence inserted into a targetingconstruct to facilitate selection of cells (e.g., ES cells) that haveintegrated the construct of interest. A number of suitable selectioncassettes are known in the art. Commonly, a selection cassette enablespositive selection in the presence of a particular antibiotic (e.g.,Neo, Hyg, Pur, CM, Spec, etc.). In addition, a selection cassette may beflanked by recombination sites, which allow deletion of the selectioncassette upon treatment with recombinase enzymes. Commonly usedrecombination sites are loxP and Frt, recognized by Cre and Flp enzymes,respectively, but others are known in the art.

In one embodiment, the promoter is a human immunoglobulin variableregion gene segment promoter. In a specific embodiment, the promoter isa human Vκ3-15 promoter. In one embodiment, the leader sequence is amouse leader sequence. In a specific embodiment, the mouse leadersequence is a mouse Vκ3-7 leader sequence. Exemplary embodiments of thetargeting vectors are presented in FIGS. 23B and 29B.

In one aspect, a targeting vector is provided as described above, but inplace of the 5′ mouse homology arm the human or mouse promoter isflanked 5′ with a site-specific recombinase recognition site (SRRS), andin place of the 3′ mouse homology arm the human V_(L) region is flanked3′ with an SRRS.

Also provided herein are methods of making genetically modifiednon-human animals (e.g., rodents, e.g., mice or rats) described herein.In one aspect, the method for making a genetically modified non-humananimal described herein utilizes a targeting vector, made usingVELOCIGENE® technology, introducing the construct into ES cells, andintroducing targeted ES cell clones into a mouse embryo usingVELOCIMOUSE® technology, as described in the Examples. Histidinemodifications may be introduced into the targeting vector using avariety of molecular biology techniques, e.g., site directed mutagenesisor de novo DNA synthesis. Upon completion of gene targeting, ES cells ofgenetically modified non-human animals are screened to confirmsuccessful incorporation of exogenous nucleotide sequence of interest orexpression of exogenous polypeptide. Numerous techniques are known tothose skilled in the art, and include (but are not limited to) Southernblotting, long PCR, quantitative PCT (e.g., real-time PCR usingTAQMAN®), fluorescence in situ hybridization, Northern blotting, flowcytometry, Western analysis, immunocytochemistry, immunohistochemistry,etc. In one example, non-human animals (e.g., mice) bearing the geneticmodification of interest can be identified by screening for loss ofmouse allele and/or gain of human allele using a modification of alleleassay described in Valenzuela et al. (2003) High-throughput engineeringof the mouse genome coupled with high-resolution expression analysis,Nature Biotech. 21(6):652-659. Other assays that identify a specificnucleotide or amino acid sequence in the genetically modified animalsare known to those skilled in the art.

Thus, in one embodiment, the method of generating genetically modifiednon-human animals comprises replacing an immunoglobulin light chainvariable region gene sequence in the animal with a human immunoglobulinlight chain variable region gene sequence (comprising human V_(L) andJ_(L) gene segments) wherein the human immunoglobulin variable regiongene sequence comprises a substitution of at least one non-histidinecodon with a histidine codon. In one embodiment, the substitution of atleast one non-histidine codon with a histidine codon is in thenucleotide sequence encoding a CDR region, e.g., a CDR3 region.

In one embodiment, the method of generating genetically modifiednon-human animals described herein comprises replacing an immunoglobulinlight chain variable region gene sequence in the animal with a singlerearranged human immunoglobulin light chain variable region genesequence comprising human V_(L) and J_(L) gene segment sequences,wherein the single rearranged human immunoglobulin variable region genesequence comprises a substitution of at least one non-histidine codonwith a histidine codon. In one embodiment, the substitution is in a CDRcodon. In one embodiment, the substitution is of one, two, three, four,or more CDR3 codon(s). In one embodiment, the single rearranged humanimmunoglobulin light chain variable region gene sequence is based on thehuman germline rearranged light chain variable region sequence selectedfrom Vκ1-39R5 and Vκ3-20R1. Thus, in one embodiment, where the singlerearranged human immunoglobulin light chain variable region genesequence is derived from Vκ1-39R5, replacement of at least onenon-histidine codon with histidine codon is designed to express ahistidine at positions selected from 105, 106, 108, 111, and acombination thereof. In one embodiment, where the single rearrangedhuman immunoglobulin light chain variable region gene sequence isderived from Vκ3-20κ1, replacement of at least one non-histidine codonwith a histidine codon is designed to express a histidine at positionselected from 105, 106, 107, 109, and a combination thereof.

In another embodiment, the method of generating a non-human animaldescribed herein (i.e., comprising a genetically modified immunoglobulinlight chain locus described herein) comprises modifying a genome of anon-human animal to delete or render non-functional endogenousimmunoglobulin light chain V and J segments in an immunoglobulin lightchain locus, and placing in the genome a single rearranged human lightchain variable region gene sequence comprising a substitution of atleast one non-histidine codon with a histidine codon. In one embodiment,the method results in a genetically modified non-human animal thatcomprises a population of B cells enriched for antibodies exhibiting pHdependent binding to an antigen of interest.

In some embodiments, the methods of generating genetically modifiednon-human animals described herein comprise replacing an immunoglobulinlight chain variable region gene sequence with human sequence in theanimal that also comprises a replacement of endogenous non-humanimmunoglobulin heavy chain variable region gene sequence with a humanimmunoglobulin heavy chain variable region gene sequence comprising atleast one of each or a repertoire of human V_(H), D_(H), and J_(H)sequences, as described above. In one embodiment, in order to generate anon-human animal comprising a replacement of endogenous immunoglobulinlight chain variable region gene sequence human light chain variableregion gene sequence comprising a substitution of at least onenon-histidine codon with a histidine codon and a replacement ofendogenous non-human immunoglobulin heavy chain variable region genesequence with a human immunoglobulin heavy chain variable region genesequence, the animal with replacement of light chain variable regiongene sequence is bred to an animal with replacement of heavy chainvariable region gene sequence.

Inventors presently provide genetically engineered non-human animals(e.g., rodents, e.g., rats or mice) that express antigen-bindingproteins, e.g., antibodies, that comprise a universal light chain, e.g.,a human universal light chain (e.g., a light chain derived from a singlerearranged human immunoglobulin light chain variable region) thatcomprises one or more histidine modifications, wherein theantigen-binding proteins exhibit a pH-dependent antigen binding of atarget antigen. The animals are genetically engineered to include alight chain CDR3 that comprises one or more histidine modifications. Invarious embodiments, the light chain CDR3 comprises two, three, or fouror more histidine residues in a cluster.

In one embodiment, provided herein is a genetically engineered non-humananimal that comprises a population of antigen-specific antibodies thatexpress histidine residue(s) as a result of codon modifications in thelight chain variable region gene sequence, and display pH-dependentbinding of target antigen. In one embodiment, these animals comprise apopulation of B cells that are enriched for antibodies, e.g.,antigen-specific antibodies, that display pH-dependent bindingproperties (e.g., decreased dissociative half-life (t_(1/2)), at acidicpH vs neutral pH) as compared to a population of antigen-specificantibodies generated in animals that do not comprise a substitution ofat least one non-histidine codon with a histidine codon inimmunoglobulin light chain variable region described herein. In oneembodiment, the enrichment of antigen-specific antibodies displayingpH-dependent antigen binding properties generated in the geneticallyengineered animals described herein as compared to similar animals thatdo comprise histidine substitutions in light chain variable region isgreater than about 2 fold, e.g., greater than about 5 fold, e.g.,greater than about 10 fold. Thus, the genetically modified animals ofthe invention are enriched for antibodies with improved antibodyrecycling properties, which is desired in order to reducetarget-mediated clearance as well as to reduce the dose and/or dosingfrequency of a therapeutic antigen-binding protein developed based onsuch in vivo generated antibody format.

Thus, provided herein is an antigen-binding protein, generated ingenetically modified non-human animals described herein, wherein theantigen-binding protein displays pH-dependent antigen binding. In oneembodiment, the antigen-binding protein is an antibody, e.g.,antigen-specific antibody. In one embodiment, the antibody comprises alight chain which comprises a human light chain variable domain derivedfrom a rearrangement of human immunoglobulin light chain variable genesegments where at least one non-histidine codon was substituted for ahistidine codon in the germline gene sequence, and wherein the antibodyretains at least one histidine substitution in its expressed human lightchain variable domain. In one embodiment, the antibody comprises a lightchain which comprises a human light chain variable domain derived from asingle rearranged human light chain variable region gene sequence,wherein the single rearranged light chain variable region gene sequencecomprises a substitution of at least one non-histidine codon with ahistidine codon, and wherein the antibody retains at least one histidinesubstitution in its expressed light chain variable domain. In oneembodiment, the antibody comprises a light chain derived from a humanVκ1-39Jκ5 or Vκ3-20Jκ1 rearrangement, wherein the human Vκ1-39Jκ5 orVκ3-20Jκ1 gene sequence comprises a substitution of at least onenon-histidine codon with a histidine codon, and wherein the antibodyretains at least one histidine substitution in its expressed light chainvariable domain. In some embodiments, the antibody retains all orsubstantially all histidine substitutions in its expressed light chainvariable domain. In one embodiment, the substitution is of threenon-histidine codons with three histidine codons in the nucleotidesequence encoding CDR3 of the light chain variable region gene sequence,and the antibody retains all three histidine substitutions in itsexpressed light chain variable domain. In one embodiment, thesubstitution is of four non-histidine codons with four histidine codonsin the nucleotide sequence encoding CDR3 of the light chain variableregion gene sequence, and the antibody retains three or four histidinesubstitutions in its expressed light chain variable domain.

In one embodiment, the light chain of the antibody further comprises anon-human light chain constant region amino acid sequence, e.g.,endogenous light chain constant region amino acid sequence. In addition,the antibody, e.g., antigen-specific antibody, generated in agenetically modified non-human animal described herein also comprises aheavy chain which comprises a human heavy chain variable domain derivedfrom a rearrangement of human heavy chain V, D, and J segments. Humanheavy chain V, D, and J segments may be selected from a repertoire ofhuman heavy chain segments present at the endogenous non-human heavychain locus, e.g., at least one functional V, at least one functional D,and at least one functional J segment, e.g., up to a complete repertoireof functional human V, D, and J segments. Exemplary possiblerearrangements of human heavy chain variable segments may be gleanedfrom a listing of functional human V, D, and J segments in IMGTdatabase, and from U.S. Application Publication Nos. 2011/0195454,2012/0021409, 2012/0192309, and 2013/0045492, incorporated herein byreference. Furthermore, in one embodiment, the heavy chain of theantibody comprises a non-human heavy chain constant region amino acidsequence, e.g., an endogenous non-human heavy chain constant regionamino acid sequence. In one embodiment, the non-human heavy chainconstant region comprises C_(H)1, hinge, C_(H)2, and C_(H)3 domains. Inone embodiment, the antibody is an IgG, IgE, IgD, IgM, or IgA isotype.

Thus, in one embodiment, provided herein is a binding protein generatedin the genetically modified non-human animals described herein, whereinthe binding protein comprises a reverse chimeric light chain comprising(a) a light chain variable domain derived from a human Vκ1-39Jκ5rearrangement comprising a substitution of at least one non-histidinecodon with a histidine codon, wherein the light chain retains at leastone histidine substitution in its expressed light chain variable domainand (b) a non-human, e.g., a mouse, light chain constant region aminoacid sequence, wherein the light chain is associated with a reversechimeric heavy chain comprising (a) a heavy chain variable domainderived from a rearrangement of human V, D, and J segments, wherein theV, D, and J segments are selected from a repertoire of human V, D, and Jsegments present in the animal, and (b) a non-human, e.g., mouse, heavychain constant region amino acid sequence. In one embodiment, therepertoire of human V, D, and J segments comprises at least onefunctional V, at least one functional D, and at least one functional Jsegment, e.g., up to a complete repertoire of functional human V, D, andJ segments. In one embodiment, the heavy and the light chain constantdomains are endogenous heavy and light chain constant regions. In oneembodiment, the heavy and light chain variable domains are somaticallymutated domains. In one embodiment, the somatically mutated light chaindomain retains at least one histidine substitution introduced into thegermline sequence. In some embodiments, the somatically mutated lightchain domain retains all or substantially all histidine substitutionsintroduced into the germline sequence. In one embodiment, theantigen-binding protein displays pH-dependent antigen bindingproperties.

In another embodiment, provided herein is a binding protein generated inthe genetically modified non-human animals described herein, wherein thebinding protein comprises a reverse chimeric light chain comprising (a)a light chain variable domain derived from a human Vκ3-20Jκ1rearrangement comprising a substitution of at least one non-histidinecodon with a histidine codon, wherein the light chain retains at leastone histidine substitution in its expressed light chain variable domainand (b) a non-human, e.g., a mouse, light chain constant region aminoacid sequence, wherein the light chain is associated with a reversechimeric heavy chain comprising (a) a heavy chain variable domainderived from a rearrangement of human V, D, and J segments, wherein theV, D, and J segments are selected from a repertoire of human V, D, and Jsegments present in the animal, and (b) a non-human, e.g., mouse, heavychain constant region amino acid sequence. In one embodiment, therepertoire of human V, D, and J segments comprises at least onefunctional V, at least one functional D, and at least one functional Jsegment, e.g., up to a complete repertoire of functional human V, D, andJ segments. In one embodiment, the heavy and the light chain constantregions are endogenous heavy and light chain constant regions. In oneembodiment, the heavy and light chain variable domains are somaticallymutated domains. In one embodiment, the somatically mutated light chaindomain retains at least one histidine substitution introduced into thegermline sequence. In some embodiments, the somatically mutated lightchain domain retains all or substantially all histidine substitutionsintroduced into the germline sequence. In one embodiment, theantigen-binding protein displays pH-dependent antigen bindingproperties.

In one embodiment, also provided herein is a B cell of the geneticallymodified animal described herein, that comprises in its germline ahistidine-modified human light chain variable region sequence, e.g., ahistidine-modified single rearranged human light chain variable regionsequence, described herein, and expresses an antigen-binding proteindescribed herein. In one embodiment, the antigen-binding protein, e.g.,an antibody, expressed in the B cell retains at least one histidineresidue introduced into the germline, and displays pH-dependentantigen-binding properties. In some embodiments, the antigen-bindingprotein, e.g., an antibody, expressed in the B cell retains all orsubstantially all histidine residues introduced into the germline, anddisplays pH-dependent antigen-binding properties.

In various embodiments, the genetically modified non-human animaldescribed herein comprises a human light chain variable region genesequence, e.g., a single rearranged human light chain variable regiongene sequence (e.g., Vκ1-39Jκ5 or Vκ3-20Jκ1 sequence) that comprises asubstitution of at least one non-histidine codon with a histidine codon(or an addition of a histidine codon into the germline sequence). Theseadditions or substitutions result in a non-human animal that comprises apopulation of B cells enriched for antigen-binding proteins with pHdependent binding properties for their antigens. In one embodiment,antigen-binding proteins, e.g., antibodies, generated in the non-humananimals described herein in response to antigen stimulation display pHdependent antigen binding while exhibiting high affinity for the antigenat neutral pH, e.g., pH between about 7.0 and about 8.0, e.g., pHbetween about 7.0 and about 7.4, e.g., between about 7.2 and about 7.4,e.g., pH of the body fluids such as blood. In one embodiment, theaffinity of the antigen-binding protein to its antigen, expressed as adissociation constant (K_(D)) at a neutral pH is less than 10⁻⁶ M, e.g.,less than 10⁻⁸M, e.g., less than 10⁻⁹ M, e.g., less than 10⁻¹⁰ M, e.g.,less than 10⁻¹¹ M, e.g., less than 10⁻¹² M.

In one embodiment, an antigen-binding protein, e.g., an antibody,generated in the genetically modified non-human animal described herein,exhibits reduced binding to its antigen in acidic pH (e.g., pH of 6.0 orlower, e.g., pH between about 5.0 and about 6.0, pH between about 5.75and about 6.0, e.g., pH of endosomal or lysosomal compartments) ascompared to neutral pH. In one embodiment, the antigen-binding protein,e.g., the antibody, generated in the genetically modified non-humananimal described herein, exhibits no binding to the antigen in acidicpH, while retaining binding to the antigen at neutral pH. In oneembodiment, an antigen-binding protein generated by the geneticallymodified non-human animal described herein, has a decrease indissociative half-life (t_(1/2)) at an acidic pH as compared to thedissociative half-life (t_(1/2)) of the antigen-binding protein at aneutral pH of at least about 2-fold, at least about 3-fold, at leastabout 4-fold, at least about 5-fold, at least about 10-fold, at leastabout 15-fold, at least about 20-fold, at least about 25-fold, or atleast about 30-fold. In one embodiment, an antigen-binding proteinexpressed by the genetically modified non-human animal described hereinhas a t_(1/2) at an acidic pH and 37° C. of about 2 min or less. In oneembodiment, an antigen-binding protein expressed by the geneticallymodified non-human animal described herein has a t_(1/2) at an acidic pHand 37° C. of less than about 1 min. In one embodiment, anantigen-binding protein expressed by the genetically modified non-humananimal described herein has a t_(1/2) at an acidic pH and 25° C. ofabout 2 min or less. In one embodiment, an antigen-binding proteinexpressed by the genetically modified non-human animal described hereinhas a t_(1/2) at an acidic pH and 25° C. of less than about 1 min.

Kinetic parameters, such as equilibrium dissociation constants (K_(D))and dissociative half-lives (t_(1/2)) can be calculated from kineticrate constant as: K_(D) (M)=k_(d) I k_(a); and t_(1/2)(min)=ln2/(60*k_(d)).

In one embodiment, the antigen-binding protein, e.g., an antibody,generated in the genetically modified non-human animals describedherein, exhibits increased binding to FcRn molecule. As described above,FcRn is a receptor present inside the endosomal compartment that iscapable of binding immunoglobulins at an acidic pH and recycling themback to the surface. Screening antibody molecules in the geneticallymodified non-human animals described herein presents a uniqueopportunity to select for antibodies with three beneficial parameters:high affinity for an antigen, pH-dependent antigen binding (with weakerantigen binding at acidic pH) and increased binding to FcRn.

In one embodiment, a genetically modified non-human animal describedherein comprises a population of B cells in response to an antigen thatproduces and is enriched for antigen-binding proteins, e.g., antibodies,that, when reformatted into therapeutics, exhibit increased serum halflife upon administration of a therapeutic dose to a subject over anequivalent B cell population produced in response to the same antigen innon-human animals that do not comprise histidine modification(s) intheir human light chain variable region gene sequences. Thus, in oneembodiment, an antigen-binding protein, e.g., an antibody, produced inresponse to an antigen of interest in a genetically modified non-humananimal described herein, when reformatted into a therapeutic, exhibitsincreased serum half life upon administration of a therapeutic dose to asubject over a serum half life of an antigen-binding protein (whenreformatted into a therapeutic and administered at the same therapeuticdose) that was produced in response to the same antigen in a non-humananimal that does not comprise histidine modification(s) in its humanlight chain variable region gene sequence. In some embodiments, theincrease in serum half life is about 2 fold, e.g., about 5 fold, e.g.,about 10 fold, e.g., about 15 fold, e.g., about 20 fold, or greater.

In one aspect, a pluripotent, induced pluripotent, or totipotent cellderived from a non-human as described herein is provided. In a specificembodiment, the cell is an embryonic stem (ES) cell.

In one aspect, a tissue derived from a non-human animal as describedherein is provided. In one embodiment, the tissue is derived fromspleen, lymph node or bone marrow of a non-human animal as describedherein.

In one aspect, a nucleus derived from a non-human animal as describedherein is provided. In one embodiment, the nucleus is from a diploidcell that is not a B cell.

In one aspect, a non-human cell is provided that is isolated from anon-human animal (e.g., a rodent, e.g., a mouse or a rat) as describedherein. In one embodiment, the cell is an ES cell. In one embodiment,the cell is a lymphocyte. In one embodiment, the lymphocyte is a B cell.In one embodiment, the B cell expresses a chimeric heavy chaincomprising a variable domain derived from a human gene segment; and alight chain derived from a rearranged human Vκ1-39/J sequence with asubstitution of at least one non-histidine codon with histidine codon,rearranged human Vκ3-20/J sequence with a substitution of at least onenon-histidine codon with histidine codon, or a combination thereof andfurther comprising a substitution of at least one amino acid encoded inthe germline for a histidine; wherein the heavy chain variable domain isfused to a non-human constant region and the light chain variable domainis fused to a non-human or a human constant region.

In one aspect, a hybridoma is provided, wherein the hybridoma is madewith a B cell of a non-human animal as described herein. In a specificembodiment, the B cell is from a mouse as described herein that has beenimmunized with an immunogen comprising an epitope of interest, and the Bcell expresses a binding protein that binds the epitope of interest, thebinding protein has a somatically mutated human variable heavy chaindomain and a mouse C_(H), and has a human variable light chain domainderived from a rearranged human Vκ1-39Jκ5 with a substitution of atleast one non-histidine codon with histidine codon or a rearranged humanVκ3-20Jκ1 with a substitution of at least one non-histidine codon withhistidine codon and a mouse C_(L), wherein the human light chain domaincomprises a substitution of at least one amino acid encoded in thegermline with a histidine.

Also provided is a cell expressing an antigen-binding protein generatedin the non-human animals described herein. In one embodiment, the cellis selected from CHO, COS, 293, HeLa, and a retinal cell expressing aviral nucleic acid sequence (e.g., a PERC.6™ cell).

In one aspect, a non-human embryo is provided, wherein the embryocomprises a donor ES cell that is derived from a non-human animal asdescribed herein.

The non-human animals described herein are useful to generate B cellsthat express antibodies having histidines in a CDR3. An animal thatplaces histidines in a CDR3 is useful for making antibodies in general,and in particular useful for developing antibodies that bind a targetwith sufficient affinity at or around a neutral pH, but that either donot bind or that bind weaker to the same target at an acidic pH.

The non-human animal is useful to generate variable regions ofantibodies that can be used to make, e.g., human therapeutic bindingproteins that bind their targets by human immunoglobulin variabledomains that comprise the histidines in a CDR3. The altered binding at alower pH will in some circumstances allow faster turnover because thetherapeutic will bind a target on a cell's surface, be internalized inan endosome, and more readily or more rapidly dissociate from the targetin the endosome, so that the therapeutic can be recycled to bind yetanother molecule of target (e.g., on another cell or the same cell). Insome circumstances, this will result in the ability to dose thetherapeutic at a lower dose, or dose the therapeutic less frequently.This is particularly useful where it is not desirable to dosefrequently, or to administer above a certain dosage, for safety ortoxicity reasons. As a result, the serum half life of the antibodytherapeutic when administered to a subject will be increased.

The non-human animal, e.g., rodent, e.g., mouse or rat, is useful in amethod for increasing the number of B cells in a animal that exhibit anantibody variable region having a CDR3 with one or more histidines init. The non-human animal is useful for generating antibody sequencesthat will exhibit pH-dependent antigen binding. The non-human animal isuseful for generating a greater number of antibody sequences, resultingfrom a single immunization, wherein the antibodies will exhibit apH-dependent antigen binding.

Antigen-Binding Proteins and Methods of Generating the Same

In one aspect, also provided herein are methods for generating humanantigen-binding proteins, e.g., antibodies, which exhibit pH-dependentantigen binding, from the genetically modified non-human animalsdescribed herein with standard methods used in the art.

Several techniques for the producing antibodies have been described. Forexample, in various embodiments chimeric antibodies are produced in miceas described herein. Antibodies can be isolated directly from B cells ofan immunized mouse (e.g., see U.S. 2007/0280945A1) and/or the B cells ofthe immunized mouse can be used to make hybridomas (Kohler and Milstein,1975, Nature 256:495-497). DNA encoding the antibodies (human heavyand/or light chains) from non-human animals as described herein isreadily isolated and sequenced using conventional techniques. Hybridomaand/or B cells derived from non-human animals as described herein serveas a preferred source of such DNA. Once isolated, the DNA may be placedinto expression vectors, which are then transfected into host cells thatdo not otherwise produce immunoglobulin protein, to obtain the synthesisof monoclonal antibodies in the recombinant host cells. The DNA also maybe modified, for example, by substituting the coding sequence for humanheavy and light chain constant domains in place of the non-humansequences. Thus, once nucleic acid sequences of antibodies with desiredcharacteristics, e.g., affinity, epitope, pH-dependent antigen binding,etc., are determined, the non-human constant region gene sequences arereplaced with a desired human constant region sequences to generate afully human antibody containing a non-IgM isotype, for example, IgG1,IgG2, IgG3 or IgG4.

Thus, in one embodiment provided herein is a method of generating anantibody that exhibits pH-dependent antigen binding propertiescomprising generating a non-human animal (e.g., a mouse) as describedherein, immunizing a mouse with an antigen of interest, allowing anon-human animal to mount an immune response to the antigen, andselecting in the non-human animal an antigen-specific antibody thatexhibits pH dependent antigen binding properties, e.g., weaker bindingto the antigen at an acidic than at neutral pH.

Also provided herein are methods of making multi-specific antigenbinding proteins, e.g., bispecific antigen-binding proteins. These aremolecules capable of binding more than one epitope with high affinity.Advantages of the invention include the ability to select suitably highbinding (e.g., affinity matured) heavy chain immunoglobulin chains eachof which will associate with a single light chain. In addition,advantages of the invention include the ability to generate amulti-specific, e.g., a bispecific, antigen-binding protein thatexhibits pH-dependent antigen binding.

Because of the dual nature of bispecific antibodies (i.e., may bespecific for different epitopes of one polypeptide or may containantigen-binding domains specific for more than one target polypeptide,see, e.g., Tutt et al., 1991, J. Immunol. 147:60-69; Kufer et al., 2004,Trends Biotechnol. 22:238-244), they offer many useful advantages fortherapeutic application. For example, the bispecific antibodies can beused for redirected cytotoxicity (e.g., to kill tumor cells), as avaccine adjuvant, for delivering thrombolytic agents to clots, forconverting enzyme activated prodrugs at a target site (e.g., a tumor),for treating infectious diseases, targeting immune complexes to cellsurface receptors, or for delivering immunotoxins to tumor cells.

The bispecific antibodies described herein can also be used in severaltherapeutic and non-therapeutic and/or diagnostic assay methods, suchas, enzyme immunoassays, two-site immunoassays, in vitro or in vivoimmunodiagnosis of various diseases (e.g., cancer), competitive bindingassays, direct and indirect sandwich assays, and immunoprecipitationassays. Other uses for the bispecific antibodies will be apparent tothose skilled in the art.

Several techniques for making bispecific antibody fragments fromrecombinant cell culture have been reported. However, synthesis andexpression of bispecific binding proteins has been problematic, in partdue to issues associated with identifying a suitable light chain thatcan associate and express with two different heavy chains, and in partdue to isolation issues. In various embodiments, compositions andmethods described herein provide the advantage of full length bispecificantibodies that do not require special modification(s) to maintaintraditional immunoglobulin structure by increasing stability/interactionof the components. In various embodiments, such modification(s) hasproven cumbersome and served as an obstacle to development of bispecificantibody technology and their potential use in treating for humandisease. Thus, in various embodiments, through providing a naturalimmunoglobulin structure (i.e., full length) having the added propertyof multiple specificities, full length bispecific antibodies maintaintheir critical effector functions that previous bispecific fragmentslacked, and further provide therapeutics that demonstrate the importantpharmacokinetic parameter of a longer half-life.

Methods and compositions described herein allow for a geneticallymodified mouse to select, through otherwise natural processes, asuitable light chain that can associate and express with more than oneheavy chain, including heavy chains that are somatically mutated (e.g.,affinity matured), wherein the light chain further confers upon theantigen-binding protein its pH-dependent antigen binding property. Humanheavy and light chain variable region sequences from suitable B cells ofimmunized mice as described herein that express affinity maturedantibodies having reverse chimeric heavy chains (i.e., human variableand mouse constant) can be identified and cloned in frame in anexpression vector with a suitable human constant region gene sequence(e.g., a human IgG1). Two such constructs can be prepared, wherein eachconstruct encodes a human heavy chain variable domain that binds adifferent epitope. One of the human light chain variable regions (e.g.,human Vκ1-39Jκ5 or human Vκ3-20Jκ1), comprising a substitution of atleast one non-histidine codon with a histidine codon, can be fused inframe to a suitable human light chain constant region gene (e.g., ahuman κ constant gene). These three fully human heavy and lightconstructs can be placed in a suitable cell for expression. The cellwill express two major species: a homodimeric heavy chain with theidentical light chain, and a heterodimeric heavy chain with theidentical light chain. To allow for a facile separation of these majorspecies, one of the heavy chains is modified to omit a Protein A-bindingdeterminant, resulting in a differential affinity of a homodimericbinding protein from a heterodimeric binding protein. Compositions andmethods that address this issue are described in U.S. Ser. No.12/832,838, filed 25 Jun. 2010, entitled “Readily Isolated BispecificAntibodies with Native Immunoglobulin Format,” published as US2010/0331527A1, hereby incorporated by reference. Once the speciecomprising heterodimeric heavy chain with an identical light chain isselected, this bi-specific antigen binding protein can be screened toconfirm the retention of its pH-dependent antigen binding property.

In one aspect, an epitope-binding protein as described herein isprovided, wherein human light chain and heavy chain variable regionsequences are derived from animals described herein that have beenimmunized with an antigen comprising an epitope of interest.

In one embodiment, an epitope-binding protein is provided that comprisesa first and a second polypeptide, the first polypeptide comprising, fromN-terminal to C-terminal, a first epitope-binding region thatselectively binds a first epitope, followed by a constant region thatcomprises a first C_(H)3 region of a human IgG selected from IgG1, IgG2,IgG4, and a combination thereof; and, a second polypeptide comprising,from N-terminal to C-terminal, a second epitope-binding region thatselectively binds a second epitope, followed by a constant region thatcomprises a second C_(H)3 region of a human IgG selected from IgG1,IgG2, IgG4, and a combination thereof, wherein the second C_(H)3 regioncomprises a modification that reduces or eliminates binding of thesecond C_(H)3 domain to protein A. Various such modifications aredescribed in, e.g., U.S. Application Publication Nos. 2010/0331527 and2011/0195454, incorporated herein by reference.

One method for making an epitope-binding protein that binds more thanone epitope and exhibits pH-dependent epitope binding property is toimmunize a first mouse in accordance with the invention with an antigenthat comprises a first epitope of interest, wherein the mouse comprises(1) an endogenous immunoglobulin light chain variable region locus thatdoes not contain an endogenous mouse light chain variable region genesequence that is capable of rearranging and forming a light chain,wherein at the endogenous mouse immunoglobulin light chain variableregion locus is a single rearranged human light chain variable regionoperably linked to the mouse endogenous light chain constant regiongene, and the rearranged human light chain variable region is selectedfrom a human Vκ1-39R5 and a human Vκ3-20R1 comprising a substitution ofat least one non-histidine codon with a histidine condon, and (2) theendogenous mouse V_(H) gene segments have been replaced in whole or inpart with human V_(H) gene segments, such that immunoglobulin heavychains made by the mouse are solely or substantially heavy chains thatcomprise human variable domains and mouse constant domains. Whenimmunized, such a mouse will make a reverse chimeric antibody,comprising only one of two human light chain variable domains (e.g., oneof human Vκ1-39Jκ5 or human Vκ3-20R1, e.g., comprising a substitution ofat least one amino acid with a histidine). Commonly, at least some ofthe substituted histidine residues introduced into the germline sequencewill be retained in the reverse chimeric antibody. Once a B cell isidentified that encodes a heavy chain variable domain that binds theepitope of interest and expresses an antibody that exhibits pH-dependentantigen binding properties, the nucleotide sequence of the heavy chainvariable region (and, optionally, the light chain variable region) canbe retrieved (e.g., by PCR) and cloned into an expression construct inframe with a suitable human immunoglobulin heavy chain constant regionsequence. This process can be repeated to identify a second heavy chainvariable domain that binds a second epitope, and a second heavy chainvariable region gene sequence can be retrieved and cloned into anexpression vector in frame to a second suitable human immunoglobulinheavy chain constant region sequence. The first and the secondimmunoglobulin constant domains encoded by the constant region genesequence can be the same or different isotype, and one of theimmunoglobulin constant domains (but not the other) can be modified asdescribed herein or in US 2010/0331527A1, and epitope-binding proteincan be expressed in a suitable cell and isolated based on itsdifferential affinity for Protein A as compared to a homodimericepitope-binding protein, e.g., as described in US 201010331527A1.

Thus, in various embodiments, following isolation of the DNA andselection of the first and second nucleic acid sequences that encode thefirst and second human heavy chain variable domains having the desiredspecificities/affinities, and a third nucleic acid sequence that encodesa human light chain domain (a germline rearranged sequence or a lightchain sequence isolated from a non-human animal as described herein) andcomprises a substitution of at least one non-histidine codon with ahistidine codon, the three nucleic acids sequences encoding themolecules are expressed to form the bispecific antibody usingrecombinant techniques which are widely available in the art. Often, theexpression system of choice will involve a mammalian cell expressionvector and host so that the bispecific antibody is appropriatelyglycosylated (e.g., in the case of bispecific antibodies comprisingantibody domains which are glycosylated). However, the molecules canalso be produced in the prokaryotic expression systems. Normally, thehost cell will be transformed with DNA encoding both the first humanheavy chain variable domain, the second human heavy chain variabledomain, the human light chain domain on a single vector or independentvectors. However, it is possible to express the first human heavy chainvariable domain, second human heavy chain variable domain, and humanlight chain domain (the bispecific antibody components) in independentexpression systems and couple the expressed polypeptides in vitro. Invarious embodiments, the human light chain domain derived from agermline sequence but for the substitution of at least one non-histidinecoding with a histidine codon, e.g., in a CDR codon. In variousembodiments, the human light chain domain comprises no more than one, nomore than two, no more than three, no more than four, or no more thanfive somatic hypermutations within the light chain variable sequence ofthe light chain domain. In some embodiments, the somatic hypermutationsdo not alter the presence of at least one histidine residue introducedinto the germline sequence of the light chain variable region.

In various embodiments, the nucleic acid(s) (e.g., cDNA or genomic DNA)encoding the two heavy chains and single human light chain with asubstitution of at least one non-histidine with a histidine is insertedinto a replicable vector for further cloning (amplification of the DNA)and/or for expression. Many vectors are available, and generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more marker genes, anenhancer element, a promoter, and a transcription termination sequence.Each component may be selected individually or based on a host cellchoice or other criteria determined experimentally. Several examples ofeach component are known in the art.

Expression and cloning vectors usually contain a promoter that isrecognized by the host organism and is operably linked to the nucleicacid sequences that encode each or all the components of the bispecificantibody. A large number of promoters recognized by a variety ofpotential host cells are well known. These promoters are operably linkedto bispecific antibody-encoding DNA by removing the promoter from thesource DNA by restriction enzyme digestion and inserting the isolatedpromoter sequence into the vector.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) may also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding the bispecific antibody components.Suitable expression vectors for various embodiments include those thatprovide for the transient expression in mammalian cells of DNA encodingthe bispecific antibody. In general, transient expression involves theuse of an expression vector that is able to replicate efficiently in ahost cell, such that the host cell accumulates many copies of theexpression vector and, in turn, synthesizes high levels of a desiredpolypeptide encoded by the expression vector. Transient expressionsystems, comprising a suitable expression vector and a host cell, allowfor the convenient positive identification of polypeptides encoded bycloned DNAs, as well as for the rapid screening of bispecific antibodieshaving desired binding specificities/affinities or the desired gelmigration characteristics relative to the parental antibodies havinghomodimers of the first or second human heavy chain variable domains.

In various embodiments, once the DNA encoding the components of thebispecific antibody are assembled into the desired vector(s) asdescribed above, they are introduced into a suitable host cell forexpression and recovery. Transfecting host cells can be accomplishedusing standard techniques known in the art appropriate to the host cellselected (e.g., electroporation, nuclear microinjection, bacterialprotoplast fusion with intact cells, or polycations, e.g., polybrene,polyornithine, etc.).

A host cell is chosen, in various embodiments, that best suits theexpression vector containing the components and allows for the mostefficient and favorable production of the bispecific antibody species.Exemplary host cells for expression include those of prokaryotes andeukaryotes (single-cell or multiple-cell), bacterial cells (e.g.,strains of E. coli, Bacillus spp., Streptomyces spp., etc.),mycobacteria cells, fungal cells, yeast cells (e.g., S. cerevisiae, S.pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells(e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni,etc.), non-human animal cells, human cells, or cell fusions such as, forexample, hybridomas or quadromas. In various embodiments, the cell is ahuman, monkey, ape, hamster, rat, or mouse cell. In various embodiments,the cell is eukaryotic cell selected from CHO (e.g., CHO K1, DXB-11 CHO,Veggie-CHO), COS (e.g., COS-7), retinal cell, Vero, CV1, kidney (e.g.,HEK293, 293 EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5,Colo205, HB 8065, HL-60, (e.g., BHK21), Jurkat, Daudi, A431 (epidermal),CV-1, U937, 3T3, L cell, C127 cell, SP2/0, NS-0, MMT 060562, Sertolicell, BRL 3A cell, HT1080 cell, myeloma cell, tumor cell, and a cellline derived from an aforementioned cell. In various embodiments, thecell comprises one or more viral genes, e.g. a retinal cell thatexpresses a viral gene (e.g., a PER.C6™ cell).

Mammalian host cells used to produce the bispecific antibody may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. Media may be supplemented asnecessary with hormones and/or other growth factors (such as insulin,transferrin, or epidermal growth factor), salts (such as sodiumchloride, calcium, magnesium, and phosphate), buffers (such as HEPES),nucleosides (such as adenosine and thymidine), antibiotics (such asGENTAMYCIN™), trace elements (defined as inorganic compounds usuallypresent at final concentrations in the micromolar range), and glucose oran equivalent energy source. Any other supplements may also be includedat appropriate concentrations as known to those skilled in the art. Theculture conditions, such as temperature, pH, and the like, are, invarious embodiments, those previously used with the host cell selectedfor expression, and will be apparent to those skilled in the art.

The bispecific antibody may be recovered from the culture medium as asecreted polypeptide, although it also may be recovered from host celllysate when directly produced without a secretory signal. If thebispecific antibody is membrane-bound, it can be released from themembrane using a suitable detergent solution (e.g., Triton-X 100).

Following isolation, a bispecific antibody comprising a two human heavychains and a single human light chain derived from a rearranged humanlight chain variable region gene sequence selected from Vκ1-39Jκ5 andVκ3-20Jκ1 sequences that comprise a substitution of at least onenon-histidine codon with a histidine codon, is screened for its abilityto exhibit pH dependent binding of one, preferably both of its antigens.The ability of bispecific antibodies to bind its antigens differently atneutral and acidic pH's (e.g., their ability to demonstrate decreasedt_(1/2) at acidic pH compared to neutral pH) can be determined by avariety of techniques available in the art and described in thefollowing examples, e.g., BIACORE™ assay.

Additional Methods for Generating Antigen-Binding Proteins withpH-Dependent Antigen Binding

Various methods of generating antigen-binding proteins with pH-dependentantigen binding properties in genetically modified non-human animalsdescribed herein are provided. Also provided are methods of generatingantigen binding proteins with pH-dependent antigen binding properties invitro. Such methods may involve generating various components of theantigen-binding proteins in vivo in genetically modified non-humananimals, and then modifying them and reassembling them in vitro outsidean organism as protein complexes expressed in mammalian cell culture.

In one embodiment, the method of generating antigen-binding proteinswith pH-dependent antigen binding properties utilizes an antigen-bindingprotein sequence, e.g., an antibody sequence, that is generated in amouse comprising a limited repertoire of light chain variable region Vand J segments, e.g., human light chain variable region V and Jsegments, “universal light chain” or “common light chain” mouse (“ULC”mouse), such as the mouse described in U.S. Application Publication Nos.2011/0195454, 2012/0021409, 2012/0192300 and 2013/0045492, allincorporated herein by reference. In one embodiment, the method ofgenerating antigen-binding proteins with pH-dependent antigen bindingproperties utilizes an antigen binding protein sequence that isgenerated in a mouse comprising a single rearranged human light chainvariable region gene sequence. In one embodiment, the method utilizes anantigen binding protein generated in a mouse comprising a singlerearranged human light chain variable region gene sequence selected fromhuman Vκ1-39Jκ5 and human Vκ3-20Jκ1.

In one embodiment, the method for generating an antigen-binding protein,e.g., an antibody, with pH dependent antigen binding propertiescomprises selecting a first antibody that binds to an antigen ofinterest (e.g., binds to an antigen of interest with a desiredaffinity), modifying an immunoglobulin light chain nucleotide sequenceof the first antibody to comprise a substitution of at least onenon-histidine codon with a histidine codon, expressing an immunoglobulinheavy chain of the first antibody and the modified immunoglobulin lightchain in a cells, and selecting a second antibody expressed in the cellthat retains binding to the antigen of interest (e.g., retains desiredaffinity for the antigen of interest) at neutral pH and displays reducedbinding to the antigen of interest at an acidic pH.

In one embodiment, the method for generating an antigen-binding protein,e.g., an antibody, with pH dependent antigen binding propertiescomprises selecting an immunoglobulin heavy chain from an antibody(e.g., obtained from a non-human animal, e.g., a mouse, e.g., a ULCmouse) that comprises an immunoglobulin light chain having a singlerearranged human immunoglobulin light chain variable region sequencewherein the antibody binds to an antigen of interest (e.g., binds to anantigen of interest with a desired affinity); modifying the nucleic acidsequence of the immunoglobulin light chain such that the singlerearranged human immunoglobulin light chain variable region sequencecomprises a substitution of at least one non-histidine codon with ahistidine codon; expressing the selected immunoglobulin heavy chain andthe immunoglobulin light chain comprising the substitution of at leastone amino acid with a histidine in its variable domain; and selecting anantibody that retains binding to the antigen of interest at a neutral pH(e.g., retains desired affinity to the antigen of interest) whiledisplaying reduced binding to the antigen of interest at an acidic pH.In various embodiments, the immunoglobulin heavy chain is derived from arearrangement of human heavy chain variable gene segments (human V, D,and J segments).

In one embodiment, the method for generating an antigen-binding protein,e.g., an antibody, with pH-dependent antigen binding propertiescomprises (1) immunizing a non-human animal, e.g., a mouse, comprising asingle rearranged human light chain variable region gene sequence and arepertoire of unrearranged human heavy chain variable gene segments (V,D, and J segments) with an antigen of interest and allowing a mouse tomount an immune response to said antigen, (2) selecting in the non-humananimal, e.g., in the mouse, an antibody that binds to the antigen ofinterest with a desired affinity, (3) isolating from the non-humananimal, e.g., from the mouse, a nucleotide sequence of an immunoglobulinheavy chain of the antibody that binds to the antigen of interest with adesired affinity, (4) determining the nucleotide sequence of said heavychain, (5) modifying a nucleotide sequence of an immunoglobulin lightchain containing the single rearranged human immunoglobulin light chainvariable region to comprise a substitution of at least one non-histidinecodon with a histidine codon, (6) expressing the immunoglobulin heavychain of the antibody that binds to the antigen of interest with desiredaffinity and the immunoglobulin light chain comprising the histidinemodification in a cell, and (7) determining whether the antibodyexpressed in the cell retains binding to the antigen at a neutral pHwhile displaying reduced binding at an acidic pH. In one embodiment, theantibody expressed in the cell exhibits desired affinity to the antigenat neutral pH. In various embodiments, the immunoglobulin heavy chain isderived from a rearrangement of human heavy chain variable gene segments(human V, D, and J segments).

In one embodiment, the mouse comprising a single rearranged human lightchain variable region gene sequence is a universal light chain or commonlight chain “ULC” mouse described in, e.g., U.S. Application PublicationNos. 2011/0195454, 2012/0021409, 2012/0192300 and 2013/0045492. In oneembodiment, the single rearranged human light chain variable region genesequence is selected from human Vκ1-39Jκ5 and human Vκ3-20Jκ1 sequence.

In one embodiment, the antigen of interest is selected from a solubleantigen, a cell surface antigen (e.g., a tumor antigen) and a cellsurface receptor. In a specific embodiment, the cell surface receptor isan immunoglobulin receptor. In a specific embodiment, the immunoglobulinreceptor is an Fc receptor.

In one embodiment, the desired affinity of an antibody for an antigenexpressed as a dissociation constant (K_(D)) at a neutral pH is lessthan 10⁻⁶ M, e.g., less than 10⁻⁸M, e.g., less than 10⁻⁹ M, e.g., lessthan 10¹⁰ M, e., g less than 10⁻¹¹ M, e.g., less than 10⁻¹² M.

As explained above, the ULC mice, in one embodiment, comprise a singlerearranged human immunoglobulin light chain variable gene sequence, andexpress antibodies in response to the antigen where the affinity ofantibodies to the antigen is primarily mediated through the heavy chainsof their antibodies. These mice comprise a repertoire of human heavychain variable (V, D, and J) segments, that rearrange to encode a humanheavy chain variable domain of an antibody that also comprises the lightchain derived from the single rearranged human light chain variablesequence. In one embodiment, upon antigen exposure, these mice utilizethe diverse repertoire of human heavy chain variable (V, D, and J)segments to generate an antibody with affinity to and specificity forthe antigen. Thus, upon exposure to the antigen, the nucleotide sequenceof an immunoglobulin heavy chain of the antibody generated in the ULCmice may be isolated and utilized to generate a desired binding proteinalso comprising an immunoglobulin light chain derived from the singlerearranged human immunoglobulin light chain variable region sequence(e.g., the single rearranged human immunoglobulin light chain variableregion sequence with a substitution of at least one non-histidine codonwith a histidine codon).

In one embodiment of the ULC mice, 90-100% of unrearranged non-humanV_(H) gene segments are replaced with at least one unrearranged humanV_(H) gene segment. In a specific embodiment, all or substantially all(e.g., 90-100%) of the endogenous non-human V_(H) gene segments arereplaced with at least one unrearranged human V_(H) gene segment. In oneembodiment, the replacement is with at least 19, at least 39, or atleast 80 or 81 unrearranged human V_(H) gene segments. In oneembodiment, the replacement is with at least 12 functional unrearrangedhuman V_(H) gene segments, at least 25 functional unrearranged humanV_(H) gene segments, or at least 43 functional unrearranged human V_(H)gene segments. In one embodiment, the non-human animal comprises areplacement of all non-human D_(H) and J_(H) segments with at least oneunrearranged human D_(H) segment and at least one unrearranged humanJ_(H) segment. In one embodiment, the non-human animal comprises areplacement of all non-human D_(H) and J_(H) segments with allunrearranged human D_(H) segments and all unrearranged human J_(H)segments. Thus, the ULC mouse utilizes a diverse repertoire of humanvariable region gene segments (V, D, and J segments) to generate anantibody in response to the antigen of interest.

Once the heavy chain of the antibody that binds to the antigen ofinterest with the desired affinity is determined, the nucleotidesequence of the heavy chain is isolated and sequenced. The sequence iscloned into a vector for expression in suitable host cells, e.g.,eukaryotic cells, e.g., CHO cells. In one embodiment, the sequence of ahuman heavy chain constant region is cloned downstream of the humanheavy chain variable region sequence isolated from the mouse (e.g., theULC mouse).

In one embodiment, the generating an antigen-binding protein withpH-dependent antigen-binding properties comprises modifying a nucleotidesequence the immunoglobulin light chain, particularly the sequence ofthe single rearranged human immunoglobulin light chain variable region,to comprise a substitution of at least one non-histidine codon with ahistidine codon. Various techniques for modifying a nucleotide sequenceare known in the art, e.g., site directed mutagenesis. In addition, anucleotide sequence comprising the desired histidine substitution may besynthesized de novo.

In one embodiment, the substitution of at least one non-histidine codonwith a histidine codon comprises a substitution resulting in expressionof one, two, three, four, or more histidine residues. In one embodiment,the substitution(s) results in expression of three or four histidineresidues. In one embodiment, the substitution(s) is in theimmunoglobulin light chain variable region. In one embodiment, thesubstitution(s) is in the CDR codon, e.g., CDR1, CDR3, and/or CDR3. Inone embodiment, the substitution(s) is in the CDR3 codon.

In one embodiment, wherein the immunoglobulin light chain nucleic acidsequence comprises Vκ1-39Jκ5 gene sequence, and the substitution(s) isin the CDR3 codon, the substitution results in expression of histidineat position selected from 105, 106, 108, 111, and combinations thereof.In one embodiment, the substitutions result in expression of histidinesat positions 105, 106, 108, and 111. In one embodiment, thesubstitutions result in expression of histidines at positions 105 and106. In one embodiment, the substitutions result in expression ofhistidines at positions 105 and 108. In one embodiment, thesubstitutions result in expression of histidines at positions 105 and111. In one embodiment, the substitutions result in expression ofhistidines at positions 106 and 108. In one embodiment, thesubstitutions result in expression of histidines at positions 106 and111. In one embodiment, the substitutions result in expression ofhistidines at positions 108 and 111. In one embodiment, thesubstitutions result in expression of histidines at positions 105, 106,and 108. In one embodiment, the substitutions result in expression ofhistidines at positions 105, 106, and 111. In one embodiment, thesubstitutions result in expression of histidines at positions 105, 108,and 111. In one embodiment, the substitutions result in expression ofhistidines at positions 106, 108, and 111. Amino acid and nucleic acidsequences of Vκ1-39Jκ5 CDR3 regions comprising various histidinesubstitutions are depicted in FIG. 16 and included in the sequencelisting.

In one embodiment, wherein the immunoglobulin light chain nucleic acidsequence comprises Vκ3-20Jκ1 gene sequence, and the substitution(s) isin the CDR3 codon, the substitution results in expression of histidineat position selected from 105, 106, 107, 109, and combinations thereof.In one embodiment, the substitutions result in expression of histidinesat positions 105, 106, 107, and 109. In one embodiment, thesubstitutions result in expression of histidines at positions 105 and106. In one embodiment, the substitutions result in expression ofhistidines at positions 105 and 107. In one embodiment, thesubstitutions result in expression of histidines at positions 105 and109. In one embodiment, the substitutions result in expression ofhistidines at positions 106 and 107. In one embodiment, thesubstitutions result in expression of histidines at positions 106 and109. In one embodiment, the substitutions result in expression ofhistidines at positions 107 and 109. In one embodiment, thesubstitutions result in expression of histidines at positions 105, 106,and 107. In one embodiment, the substitutions result in expression ofhistidines at positions 105, 106, and 109. In one embodiment, thesubstitutions result in expression of histidines at positions 105, 107,and 109. In one embodiment, the substitutions result in expression ofhistidines at positions 106, 107, and 109. Selected amino acid andnucleic acid sequences of Vκ3-20Jκ1 CDR3 regions comprising varioushistidine substitutions are depicted in FIG. 27 and included in thesequence listing.

Once the sequence of immunoglobulin light chain, e.g., humanimmunoglobulin light chain variable domain, is modified to includehistidine residues at desired positions, the nucleotide sequence of thelight chain is cloned into a vector for expression in suitable hostcells, e.g., eukaryotic cells, e.g., CHO cells. In one embodiment, thesequence of a human light chain constant region is cloned downstream ofthe modified nucleotide sequence of human variable region.

In one embodiment, vectors comprising nucleotide sequence encodingmodified human immunoglobulin light chain and selected humanimmunoglobulin heavy chain are co-expressed in a suitable host cell,e.g., eukaryotic host cell, e.g., CHO cell, to generate anantigen-binding protein. Various host cells that can be used forexpression are known in the art and are mentioned throughout thisspecification.

An antigen-binding protein, e.g., an antibody, generated in the hostcell may be secreted into cell supernatant, which is screened for properexpression and affinity for the original antigen at neutral pH. Theantigen-binding protein may also be recovered from cell lysate, or, ifmembrane bound, released from the membrane using a suitable detergent(e.g., Triton-X). The antigen-binding protein with desiredcharacteristics may be purified.

In one embodiment, the antigen-binding protein comprising histidinemodification(s) retains the affinity to the antigen that is comparableto the affinity to the antigen of the same (original) antigen-bindingprotein that does not comprise histidine modification(s). In oneembodiment, the affinity of the histidine-modified antigen-bindingprotein for the antigen of interest expressed as a dissociation constant(K_(D)) at a neutral pH is less than 10⁻⁶ M, e.g., less than 10⁻⁸M,e.g., less than 10⁻⁹ M, e.g., less than 10⁻¹⁰ M, e.g., less than 10⁻¹¹M, e.g., less than 10⁻¹² M.

In one embodiment, the antigen-binding protein, e.g., an antibody,comprising histidine modifications described herein exhibits pHdependent antigen binding properties. In one embodiment, theantigen-binding protein comprising histidine modifications possessesenhanced pH dependent properties over an equivalent antigen-bindingprotein without the histidine modifications (antigen-binding protein ofthe same amino acid sequence but for the histidine modifications). Inone embodiment, the antigen-binding protein described herein retainsbinding to the antigen at neutral pH (e.g., retains desired affinity forthe antigen at neutral pH) while displaying reduced binding at an acidicpH. In one embodiment, the antigen-binding protein, e.g., the antibody,described herein, exhibits no binding to the antigen in acidic pH, whileretaining binding to the antigen at neutral pH. In one embodiment, anantigen-binding protein described herein, has a decrease in dissociativehalf-life (t_(1/2)) at an acidic pH as compared to the dissociativehalf-life (t_(1/2)) of the antigen-binding protein at a neutral pH of atleast about 2-fold, at least about 3-fold, at least about 4-fold, atleast about 5-fold, at least about 10-fold, at least about 15-fold, atleast about 20-fold, at least about 25-fold, or at least about 30-fold.In one embodiment, an antigen-binding protein described herein has at_(1/2) at an acidic pH and 37° C. of about 2 min or less. In oneembodiment, an antigen-binding protein described herein has a t_(1/2) atan acidic pH and 37° C. of less than about 1 min. In one embodiment, anantigen-binding protein described herein has a t_(1/2) at an acidic pHand 25° C. of about 2 min or less. In one embodiment, an antigen-bindingprotein described herein has a t_(1/2) at an acidic pH and 25° C. ofless than about 1 min.

In one embodiment, the antigen-binding protein e.g., the antibody,comprising histidine modifications described herein, exhibits increasedserum half life upon administration of a therapeutic dose to a subjectas compared to a serum half life upon administration of an equivalenttherapeutic dose of antigen-binding protein that does not comprisehistidine modifications (e.g., the original antigen-binding protein thatdoes not comprise histidine modifications). In some embodiments, theincrease in serum half life upon administration of a dose of theantigen-binding protein comprising histidine modifications describedherein over a serum half life upon administration of the same dose ofthe antigen-binding protein not comprising histidine modifications isabout 2 fold, e.g., about 5 fold, e.g., about 10 fold, e.g., about 15fold, e.g., about 20 fold, or greater. In one embodiment, serumhalf-life is at least about 1 day, e.g., at least about 2 days, e.g., atleast about 7 days, e.g., at least about 14 days, e.g., at least about30 days, e.g., at least about 60 days.

In addition to the in vitro methods for generating antigen-bindingproteins with pH-dependent antigen binding properties described above,also provided herein are antigen-binding proteins, e.g., antibodies,generated by said method. In addition, said method may be utilized togenerate multi-specific, e.g., bispecific, antigen-binding proteins, byselecting two different human immunoglobulin heavy chains that bind to acommon (universal) light chain in a mouse, determining nucleotidesequences of the heavy chains, modifying universal light chain tocomprise histidine substitutions as described above, and co-expressingtwo human heavy chains with a single histidine-modified universal lightchain in a host cell. Various steps for generating an antigen-bindingprotein described above may be applicable to the method of generating abispecific antigen-binding protein. Bispecific antigen binding protein,confirmed to possess desired affinity for the antigen(s) andpH-dependent antigen binding properties may be purified. Thus,bispecific antibodies comprising two human heavy chains and a singlehuman light chain comprising a human light chain variable domainsequence encoded by a human variable region gene, e.g., Vκ1-39Jκ5 orVκ3-20R1 variable region gene comprising a substitution of at least onenon-histidine codon with a histidine codon, is provided.

Also provided are constructs utilized in making an antigen-bindingprotein comprising human immunoglobulin heavy chain and humanimmunoglobulin light chain comprising histidine substitutions. Hostcells expressing antigen-binding proteins, e.g., antibodies, describedherein are also provided.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

Example 1 Construction of Humanized Immunoglobulin Heavy Chain LociComprising Histidine-Substituted D Gene Segments

Construction of immunoglobulin heavy chain loci comprisinghistidine-substituted human D gene segments was carried out by series ofhomologous recombination reactions in bacterial cells (BHR) usingBacterial Artificial Chromosome (BAC) DNA. Several targeting constructsfor creation of a genetically engineered mouse that expresses a heavychain variable domain comprising one or more histidine residues weregenerated using VELOCIGENE® genetic engineering technology (see, e.g.,U.S. Pat. No. 6,586,251 and Valenzuela, D. M. et al. (2003),High-throughput engineering of the mouse genome coupled withhigh-resolution expression analysis, Nature Biotechnology 21(6):652-659,incorporated herein by reference in their entireties).

Initially, human D gene segments were synthesized in silico as fourpieces (4 repeats) in which the codons encoding tyrosine (Y), asparagine(N), serine (S), glycine (G), and aspartate (D) in the hydrophilic framewere substituted with histidine codons (hereinafter“histidine-substituted human D gene segments”, i.e., HD 1.1-6.6 (9586bp; SEQ ID NO: 1), HD 1.7-6.13 (9268 bp; SEQ ID NO: 2), HD 1.14-6.19(9441 bp; SEQ ID NO: 3), and HD 1.20-6.25, 1.26 (11592 bp; SEQ ID NO: 4)(FIG. 3). The four repeats also contained unique restriction enzymesites at the ends for ligating them back together. The specific locationof the histidine substitutions (labeled in bold type) in each human Dgene segment is shown in FIGS. 1A and 1B in the column labeled“Hydrophilic.” As shown in FIG. 1, while the modification introducedhistidine codons in the hydrophilic reading frame, it also changed somestop codons to serine codons in the “Stop” reading frame. Themodification, however, made few changes in the “Hydrophobic” readingframe. The detailed procedure for ligating the four synthesized Dsegment repeats is illustrated in FIG. 3 (sequential ligation). Theresulting clone contained, from 5′ to 3′, a 5′ mouse homology arm, afloxed neomycin cassette, human D gene segments comprising histidinesubstitutions (i.e., HD 1.1-6.6 (9586 bp; SEQ ID NO: 1), HD 1.7-6.13(9268 bp; SEQ ID NO: 2), HD 1.14-6.19 (9441 bp; SEQ ID NO: 3), and HD1.20-6.25, 1.26 (11592 bp; SEQ ID NO: 4)), a chloramphenicol selectioncassette, and a 3′ homology arm.

The following six genetic modifications were carried out in order toreplace the endogenous human D gene segments in the VELOCIMMUNE®humanized mouse with the histidine-substituted human D gene segmentsdescribed above.

First, pLMa0174, containing a spectinomycin selection cassette and anAsiSI restriction site, was targeted into the 5′ end of the MAID 1116clone (Step 1. BHR (Spec); FIG. 2). During Step 1, a chloramphenicolselection cassette, a neomycin selection cassette, a loxP site, twoV_(H) gene segments (hV_(H)1-3 and hV_(H)1-2), and the human Adam6pgene, all of which are located 5′ upstream of hV_(H)6-1, were deletedfrom the MAID 1116 clone and replaced by a spectinomycin cassette toyield the VI433 clone.

Second, in Step 2 (BHR (Hyg+Spec); FIG. 2), pNTu0002 containing ahygromycin cassette flanked by FRT sites was targeted into a regioncomprising human immunoglobulin D_(H) gene segments. During Step 2, allhuman heavy chain D gene segments were deleted from VI433 and replacedwith the hygromycin cassette to yield MAID6011 VI 434 (clone 1). Themodification also introduced the PI-Scel and the I-Ceul restrictionsites at the 5′ and 3′ end of the hygromycin cassette.

Third, the genomic region comprising histidine-substituted human D genesegments (HD 1.1-6.6 (9586 bp; SEQ ID NO: 1), HD 1.7-6.13 (9268 bp; SEQID NO: 2), HD 1.14-6.19 (9441 bp; SEQ ID NO: 3), and HD 1.20-6.25, 1.26(11592 bp; SEQ ID NO: 4)) were introduced into a region between thePI-Scel and the I-Ceul sites of MAID 6011 VI434 via restrictiondigestion and ligation (PI-Scel/l-Ceul Ligation modified 1116(Kan+Spec); FIG. 4). This yielded MAID6012 VI469 containing, from 5′ to3′, a spectinomycin cassette, about 50 kb of a genomic region comprisingV_(H)6-1, a floxed neomycin cassette, about 40 kb of thehistidine-substituted human D gene segments (HD 1.1-6.6 (9586 bp; SEQ IDNO: 1), HD 1.7-6.13 (9268 bp; SEQ ID NO: 2), HD 1.14-6.19 (9441 bp; SEQID NO: 3), and HD 1.20-6.25, 1.26 (11592 bp; SEQ ID NO: 4)), and about25 kb of a genomic region containing human J_(H) gene segments, followedby a mouse E_(i) (mIgH intronic enhancer; SEQ ID NO: 5), a mouse switchregion (SEQ ID NO: 6), and a mouse IgM constant region nucleotidesequence (mIgM exon 1; SEQ ID NO: 7). Bacterial cells containing themodification were selected based on Kanamycin and Spectinomycinselection.

Fourth, MAID 1460 heterozygous mouse ES cells were targeted with MAID6011 VI434 via electroporation in order to remove all endogenous human Dgene segments from the MAID 1460 clone as illustrated in FIG. 5. Thisyielded MAID 6011 heterozygous mouse ES cells comprising in itsimmunoglobulin heavy chain locus (at the 129 strain-derived chromosome),from 5′ to 3′, an FRT site, human V_(H) gene segments, a mouse genomicregion encompassing adam6a/b genes, a hygromycin cassette flanked by FRTsites, and human J_(H) segments, followed by a mouse E, sequence and anIgM constant region nucleotide sequence. The genetic modification ofMAID 6011 (a loss of alleles, a gain of alleles, and presence ofparental alleles) was confirmed by using the probes and primers as shownin FIG. 6.

Fifth, MAID 6011 heterozygous mouse ES cells were electroporated withMAID 6012 VI469 in order to introduce histidine-substituted human D genesegments (i.e., HD 1.1-6.6 (9586 bp; SEQ ID NO: 1), HD 1.7-6.13 (9268bp; SEQ ID NO: 2), HD 1.14-6.19 (9441 bp; SEQ ID NO: 3), and HD1.20-6.25, 1.26 (11592 bp; SEQ ID NO: 4)) into MAID 6011. The targetingstep removed the floxed hygromycin selection cassette from MAID 6011 andreplaced the sequence with the histidine-substituted human D genesegments. This lead to MAID 6012 hetrozygous ES cells comprising awild-type C57BL/6 strain-derived chromosome and a genetically modified129 strain-derived chromosome comprising human wild-type V_(H) and J_(H)gene segments and the histidine-substituted human D gene segmentsdescribed herein. In addition, the ES cells contained a mouse genomicregion encompassing adam6a/b genes and a floxed neomycin cassettebetween the V_(H) and D segments (FIG. 7). The genetic modification ofMAID 6012 (a loss of alleles, a gain of alleles, and presence ofparental alleles) was confirmed by using the probes and primers as shownin FIG. 8.

Lastly, MAID 6012 ES cells were electroporated with a plasmid thatexpresses a Cre recombinase in order to remove the neomycin selectioncassette from the MAID 6012 ES cells, resulting in MAID 6013heterozygous ES cells (FIG. 9). The final MAID 6013 heterozygous (“MAID6013 het”) ES cell contains a wild-type C57BL/6 strain-derivedchromosome and a genetically modified, 129 strain-derived chromosomecomprising in its immunoglobulin heavy chain locus, from 5′ to 3′, (1)an FRT site; (2) human V_(H) gene segments; (3) a mouse genomic regionencompassing adam6a/b genes; (4) a floxed neomycin selection cassette;(5) histidine-substituted human D gene segments (HD 1.1-6.6 (9586 bp;SEQ ID NO: 1), HD 1.7-6.13 (9268 bp; SEQ ID NO: 2), HD 1.14-6.19 (9441bp; SEQ ID NO: 3), and HD 1.20-6.25, 1.26 (11592 bp; SEQ ID NO: 4)); (6)human J_(H) gene segments; followed by (7) a mouse E, sequence (mIgHintronic enhancer; SEQ ID NO: 5), (8) a switch region (SEQ ID NO: 6);and (9) a mouse IgM constant region nucleotide sequence (mIgM exon 1;SEQ ID NO: 7) as illustrated in FIG. 9.

The targeted ES cells (MAID 6013) described above were used as donor EScells and introduced into an 8-cell stage mouse embryo by theVELOCIMOUSE® method (see, e.g., U.S. Pat. Nos. 7,576,259, 7,659,442,7,294,754, US 2008-0078000 A1, all of which are incorporated byreference herein in their entireties). Mice bearing the geneticallymodified immunoglobulin heavy chain locus comprising thehistidine-substituted human heavy chain D gene segments described hereinwere identified by genotyping using the primers and probes set forth inFIG. 8. The resulting genetically modified F0 mouse was crossed to awild-type mouse to obtain F1 offspring. F1 pups were genotyped, and theF1 pups that are heterozygous for the genetically modifiedimmunoglobulin locus comprising histidine-substituted human heavy chainD gene segments were selected for further characterization.

Example 2 Analysis of Rearranged Heavy Chain Variable Region NucleotideSequences

Next, it was examined whether the genetically modified mouse comprisinghistidine-substituted human D gene segments described herein, e.g., 6013F0 heterozygous mouse, which comprises in its germline a 129strain-derived chromosome comprising human V_(H), J_(H) gene segments,and histidine-substituted human D gene segments (HD 1.1-6.6 (9586 bp;SEQ ID NO: 1), HD 1.7-6.13 (9268 bp; SEQ ID NO: 2), HD 1.14-6.19 (9441bp; SEQ ID NO: 3), and HD 1.20-6.25, 1.26 (11592 bp; SEQ ID NO: 4), canexpress rearranged heavy chain V(D)J sequences comprising one or morehistidine codons derived from the genetically modified immunoglobulinheavy chain locus.

To this end, mRNA sequences isolated from splenic B cells of the 6013 F0heterozygous mice were analyzed by reverse-transcriptase polymerasechain reaction (RT-PCR) for the presence of IgM CDR3 sequences derivedfrom the histidine-substituted human D gene segments.

Briefly, spleens were harvested and homogenized in 1×PBS (Gibco) usingglass slides. Cells were pelleted in a centrifuge (500×g for 5 minutes),and red blood cells were lysed in ACK Lysis buffer (Gibco) for 3minutes. Cells were washed with 1×PBS and filtered using a 0.7 μm cellstrainer. B-cells were isolated from spleen cells using MACS magneticpositive selection for CD19 (Miltenyi Biotec). Total RNA was isolatedfrom pelleted B-cells using the RNeasy Plus kit(Qiagen). PolyA+ mRNA wasisolated from total RNA using the Oligotex® Direct mRNA mini kit(Qiagen).

Double-stranded cDNA was prepared from splenic B cell mRNA by 5′ RACEusing the SMARTer™ Pico cDNA Synthesis Kit (Clontech). The Clontechreverse transcriptase and dNTPs were substituted with Superscript II anddNTPs from Invitrogen. Heavy chain variable region (V_(H)) antibodyrepertoires were amplified from the cDNA using primers specific for IgMconstant regions and the SMARTer™ 5′ RACE primer (Table 1). PCR productswere cleaned up using a QIAquick® PCR Purification Kit (Qiagen). Asecond round of PCR was done using the same 5′ RACE primer and a nested3′ primer specific for the IgM constant regions (Table 2). Second roundPCR products were purified using a SizeSelect™ E-gel® system(Invitrogen). A third PCR was performed with primers that added 454adapters and barcodes. Third round PCR products were purified usingAgencourt® AMPure® XP Beads. Purified PCR products were quantified bySYBR®-qPCR using a KAPA Library Quantification Kit (KAPA Biosystems).Pooled libraries were subjected to emulsion PCR (emPCR) using the 454 GSJunior Titanium Series Lib-A emPCR Kit (Roche Diagnostics) andbidirectional sequencing using Roche 454 GS Junior instrument accordingto the manufacturer's protocols.

TABLE 1  NAME SEQUENCE 3′ mIgM CH1 TCTTATCAGACAGGGGGCTCTC  outer(SEQ ID NO: 318)

TABLE 2  NAME 3′ mIgM CH1 GGAAGACATTTGGGAAGGACTG  inner (SEQ ID NO: 319)

Bioinfomatic Analysis

The 454 sequences were sorted based on the sample barcode perfect matchand trimmed for quality. Sequences were annotated based on alignment ofrearranged Ig sequences to human germline V,D and J segments databaseusing local installation of igblast (NCBI, v2.2.25+). A sequence wasmarked as ambiguous and removed from analysis when multiple best hitswith identical score were detected. A set of perl scripts was developedto analyze results and store data in mysql database. The CDR3 region wasdefined between conserved C codon and FGXG motif (SEQ ID NO: 320) forlight chains and WGXG motif (SEQ ID NO: 321) for heavy chains. CDR3length was determined using only productive antibodies.

As shown in FIGS. 11-13, 6013 F0 heterozygous mice expressed a diverserepertoire of rearranged heavy chain variable region mRNA sequences(rearranged V-D-J sequences) encoding one or more histidine codons inCDR3. The sequencing data suggested that the histidine codons appearedin CDR3 were derived from various histidine-substituted human D genesegments present in the genetically modified immunoglobulin heavy chainlocus of the 6013 mice described herein.

Example 3 Histidine Usage in Antigen-Specific Human Light Chains

Amino acid sequences of selected light chains from antigen-specifichuman antibodies were aligned. Histidine mutations in the CDRs of humanVκ1-39-derived light chains for a selected number of antigen-specifichuman antibodies were identified (FIG. 15). The human Vκ1-39-derivedlight chains were isolated from immunized mice engineered to contain asingle rearranged human Vκ1-39 light chain (see US 2011/0195454A1,herein incorporated by reference), and bear somatic hypermutations asgenerated in the antibody repertoire of the mouse.

Histidine residues were engineered into a rearranged human Vκ1-39 lightchain using molecular mutagenesis techniques known in the art. Locationsof the engineered residues are shown in FIG. 16.

Human Vκ1-39-derived light chain variable regions containing engineeredhistidine residues were constructed and paired with various human heavychain variable regions in an antibody format, specific for a human cellsurface receptor, to analyze expression in CHO cells.

CHO cells having a particular heavy chain and a light chain withindicated his modifications (e.g., 105, 106, 108, 111) were seeded intowells of a 48-well plate. The next day, DNA corresponding to heavy chainand light chain, in equal weight (400 ng), were mixed with transfectionreagent (Lipofectin 2000), allowed to form a complex by incubation, andthe complex added to the plated cells. Four days later, media wascollected. The media contained the expressed antibody.

CHO cells having different heavy chains paired with the same light chainhaving one or more his substitutions in CDR3 express well. Level (ng/mL)of antibody expression in ng/mL detected in supernatants of CHO cellstransfected with antibody genes having histidine residues engineered atselected locations in the CDR3 of the light chains was determined.

Expression in supernatants of CHO cells of paired antigen-specific heavychains with histidine engineered light chains using selected heavychains, measured by protein blots, is shown in FIG. 18. ULC refers to arearranged human Vκ1-39-derived light chain.

An aliquot of media was subjected to analysis on a BIACORE™ instrumentusing the target antigen for the antibody (a cell surface receptorsequence). Antibody was captured on the chip. Antibody capture level isshown in FIG. 19A-19J as RU. Captured antibody on the BIACORE™ chip wassubjected to flow containing the sequence of the target antigen.Antibody capture of the target antigen was measured, as well asassociation rate and other parameters as shown. Antigen flow was stoppedand dissociation rate was determined as antigen disengaged from thebound antibody.

Equilibrium dissociation constants (K_(D)) (apparent) for selectedantibody supernatants were determined by SPR (Surface Plasmon Resonance)using a BIACORE™ T100 instrument (GE Healthcare). Kinetics were measuredat pH 7.4 and at pH 5.75. Results are shown in FIG. 19A-19J.

As shown in FIG. 19A-19J, data for antibody binding to a cell surfacereceptor, where the light chains have been modified to encode histidineresidues a specific positions in a CDR, for Vκ1-30/Jκ5 light chainspaired with the indicated heavy chains, demonstrates that the histidinemodifications directly influence binding of the antigen (e.g., a cellsurface receptor) with different affinities at pH 7.4 and pH 5.75.Histidine modifications that retain binding at pH 7.4, but that exhibita low binding or no detectable binding at pH 5.75, are desirable.

Example 4 Identification of Histidine Residues in Antigen-Specific HumanLight Chains

Generation of a common light chain mouse (e.g., Vκ1-39 or Vκ3-20 commonlight chain mouse) and antigen-specific antibodies in those mice isdescribed in U.S. patent application Ser. Nos. 13/022,759, 13/093,156,and 13/412,936 (Publication Nos. 2011/0195454, 2012/0021409, and2012/0192300, respectively), incorporated by reference herein in theirentireties. Briefly, rearranged human germline light chain targetingvector was made using VELOCIGENE® technology (see, e.g., U.S. Pat. No.6,586,251 and Valenzuela et al. (2003) High-throughput engineering ofthe mouse genome coupled with high-resolution expression analysis,Nature Biotech. 21(6): 652-659) to modify mouse genomic BacterialArtificial Chromosome (BAC) clones, and genomic constructs wereengineered to contain a single rearranged human germline light chainregion and inserted into an endogenous κ light chain locus that waspreviously modified to delete the endogenous κ variable and joining genesegments. Targeted BAC DNA was then used to electroporate mouse ES cellsto create modified ES cells for generating chimeric mice that express arearranged human germline Vκ1-39Jκ5 or Vκ3-20Jκ1 region. Targeted EScells were used as donor ES cells and introduced into an 8-cell stagemouse embryo by the VELOCIMOUSE® method (see, e.g., U.S. Pat. No.7,294,754 and Poueymirou et al. (2007) F0 generation mice that areessentially fully derived from the donor gene-targeted ES cells allowingimmediate phenotypic analyses Nature Biotech. 25(1): 91-99). VELOCIMICE®independently bearing an engineered human germline Vκ1-39Jκ5 orVκ3-20Jκ1 light chain region were identified by genotyping using amodification of allele assay (Valenzuela et al., supra) that detects thepresence of the unique rearranged human germline light chain region.

Mice bearing an engineered human germline light chain locus (ULC mice)were bred with mice that contain a replacement of the endogenous mouseheavy chain variable gene locus with the human heavy chain variable genelocus (see U.S. Pat. No. 6,596,541; the VELOCIMMUNE® mouse, RegeneronPharmaceuticals, Inc.).

VELOCIMMUNE® mouse containing a single rearranged human germline lightchain region is challenged with an antigen of interest and antibodiescomprising a universal light chain (e.g., Vκ1-39Jκ5) are isolated andsequenced. Amino acid sequences of selected light chains (A-K) fromantigen-specific human antibodies generated in a common Vκ1-39Jκ5 lightchain mouse were aligned. Histidine mutations in the CDRs of humanVκ1-39-derived light chains for a selected number of antigen-specifichuman antibodies were identified (FIG. 15). The partial amino acidsequence of germline Vκ1-39Jκ5 variable domain is shown above thealignments and set forth in SEQ ID NO:325, the complete variable domainamino acid sequence is set forth in SEQ ID NO:404.

Example 5 Engineering and Characterization of Histidine-SubstitutedHuman Universal Light Chain Antibodies Example 5.1 Engineering ofHistidine Residues into a Germline Human Rearranged Light Chain

Histidine residues were engineered into a rearranged human Vκ1-39Jκ5light chain using site directed mutagenesis primers specificallydesigned to introduce engineered histidine residues at Q105, Q106, Y108,and P111 positions of the human Vκ1-39Jκ5 light chain. Site directedmutagenesis was performed using molecular techniques known in the art(e.g., QuikChange II XL Site Directed Mutagenesis Kit, AgilentTechnologies). Locations of the engineered residues in the CDR3 areshown in FIG. 16, the nucleic acid sequences of histidine-substitutedCDR3's depicted in FIG. 16 are set forth in SEQ ID NOs: 328, 330, 332,334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, and 356(corresponding amino acid sequences are set forth in SEQ ID NOs: 329,331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, and357). The nucleic acid and amino acid sequences of germline rearrangedVκ1-39R5 CDR3 are set forth in SEQ ID NOs: 326 and 327, respectively.

Example 5.2 Construction and Expression of Histidine Engineered LightChains

Human Vκ1-39-derived light chains containing germline engineeredhistidine residues made according to Example 2 were constructed andpaired with various human heavy chains (labeled 1-5), specific for ahuman cell surface receptor, to analyze expression in CHO cells. Thefive human heavy chains specific for a human cell surface receptor thatwere paired with histidine-substituted Vκ1-39-derived light chains wereobtained from mice that have a single rearranged human light chain (ahuman Vκ1-39/Jκ5 rearranged light chain; see US2011/0195454A1).

Enzyme-Linked Immunosorbent Assay (ELISA):Antibody secretion from CHOcells was detected using an Fc ELISA, for light chains with indicatedhistidine modifications with five different heavy chains. The light andheavy chain sequences (but for the modifications) were generated in micethat have a single rearranged human light chain (e.g., a humanVκ1-39/Jκ5 rearranged light chain; see US2011/0195454A1). Captureantibody was goat anti-human IgG and detection antibody was goatanti-human (Fc gamma-specific)-HRP. The results are shown in FIG. 17.ULC+heavy: specific heavy chain and unmodified human Vκ1-39-derivedlight chain. As shown in FIG. 17, expression was detected in about allmutants.

Protein Immunoblot. Expression in supernatants of CHO cells of pairedantigen-specific heavy chains with histidine engineered light chains wasfurther analyzed by western blot. Samples were run on a 4-12%tris-glycine gel. Results using a selected heavy chain (heavy chain 3)are shown in FIG. 18. ULC refers to a rearranged human Vκ1-39-derivedlight chain (as described above).

Example 5.3 Determination of Binding Affinity of Histidine EngineeredLight Chains

Equilibrium dissociation constants (K_(D)), dissociative half-lives(t_(1/2)), and other kinetic parameters for selected antibodysupernatants were determined by SPR (Surface Plasmon Resonance) using aBIACORE™ T200 instrument (GE Healthcare). Kinetics were measured at pH7.4 and at pH 5.75. Results are shown in FIGS. 20A-20E.

Numerical values for the kinetic binding properties (e.g., k_(a), k_(d),K_(D), t_(1/2), etc.) of antibodies binding to immunogen at neutral pH(pH 7.4) and at acidic pH (pH 5.75) were obtained using a real-timesurface plasmon resonance biosensor (Biacore T200.) A Biacore CM5 sensorchip was derivatized with a mouse anti-human Fc antibody to captureantibodies from the supernatant. A single concentration (50 nM) ofimmunogen was then injected over the antibody-captured surface at a flowrate of 30 μl/min. Antibody-antigen association was monitored for 2.5minutes and then the dissociation of antigen from the captured antibodywas monitored for 8 minutes. Kinetic association (ka) and dissociation(kd) rate constants were determined by processing and fitting the datato a 1:1 binding with a mass transport model using Biacore T200Evaluation software version 1.0. Equilibrium dissociation constants(K_(D)) and dissociative half-lives (t_(1/2)) were calculated from thekinetic rate constants as: K_(D) (M)=k_(d)/k_(a); and t_(1/2) (min)=(ln2/(60*k_(d)).

As shown in FIG. 20, in a binding assay of antibody to a cell surfacereceptor, two out of five antibodies with histidine-modified commonlight chains (histidine modified CDR3's of Vκ1-39/Jκ5 light chains) thatwere paired with the antigen-specific human heavy chains, exhibitedbinding to the antigen (e.g., to a cell surface receptor) with differentaffinities at pH 7.4 and pH 5.75. Antibodies with histidinemodifications that retain binding at pH 7.4, but that exhibit a lowbinding or no detectable binding at pH 5.75, are desirable. Antibodieswith histidine modification that exhibit reduced t_(1/2) at pH 5.75 ascompared to pH 7.4 are desirable.

Antigen binding data for three antibodies comprising histidine-modifiedcommon light chains and three antigen-specific heavy chains (labeled 2,3, and 6) at different pHs is summarized further in FIG. 21. Theseantibodies exhibited significant drop in antigen binding at pH 5.75 incomparison to pH 7.4, as demonstrated, e.g., by reduction in t_(1/2) orno binding detected at pH 5.75.

Example 6 Engineering and Characterization of Genetically Modified MouseComprising a Histidine-Substituted Vκ1-39Jκ5 Universal Light ChainExample 6.1 Constructing of Targeting Vector for Engineering HistidineResidues in a Rearranged Human Light Chain Variable Region

A genetically modified mouse containing a rearranged human light chaingene having histidine residues engineered into a CDR region of the humanlight chain is made using targeting vectors made by standard molecularcloning techniques known in the art.

Briefly, various rearranged human germline light chain targeting vectorsare made using VELOCIGENE® technology (see, e.g., U.S. Pat. No.6,586,251 and Valenzuela et al. (2003) High-throughput engineering ofthe mouse genome coupled with high-resolution expression analysis,Nature Biotech. 21(6):652-659) to modify mouse genomic BacterialArtificial Chromosome (BAC) DNA to contain a single rearranged humangermline light chain region and inserted into an endogenous κ lightchain locus that was previously modified to delete the endogenous κvariable and joining gene segments. The rearranged human germline lightchain region is modified at one or more nucleotide positions within thesequence of the light chain to encode histidine residues that are notnormally present at the respective locations of the germline sequence.The targeting vectors are electroporated into mouse embryonic stem (ES)cells to create and confirmed using a quantitative PCR assay (e.g.,TAQMANT™).

Specifically, a strategy for constructing these targeting vectors isshown in FIGS. 23A-23F. A plasmid used for generating a targeting vectorfor common (universal) light chain mouse (“ULC mouse,” described in,e.g., US2011/0195454A1), containing pBS+FRT-Ub-Hyg-FRT+mouse Vκ3-7leader+human Vκ1-39R5 was modified by site directed mutagenesis(QuickChange II XL Kit) to replace Q105, Q106, Y108 and P111 or Q106,Y108 and P111 with histidine residues in the CDR3 region usingsite-directed mutagenesis primers shown in FIG. 22 (See FIG. 23A forthis engineering step). Resultant vectors (H105/106/108/111 andH106/108/111) were modified further and ligated into a vector comprisingmouse Igκ constant region, mouse enhancers, a mouse 3′ homology arm anda SPEC cassette (FIG. 23B). Further modification involved ligation intoa vector carrying 5′ mouse arm and comprising Frt-Ub-NEO-Frt cassette(FIG. 23B). Resultant targeting vectors were electroporated into EScells comprising deletion of the mouse Igκ variable locus (comprising κvariable and joining gene segments) (FIGS. 23C-23F).

Positive ES cell clones were confirmed by using a modification of alleleassay (Valenzuela et al.) using probes specific for the engineeredVκ1-39R5 light chain region inserted into the endogenous κ light chainlocus. Primers and probes used in the assay are shown in Table 3 belowand set forth in the Sequence Listing; the locations of the probes aredepicted in FIGS. 23C-23F.

TABLE 3  Primers and Probes Used for ES Cell Screening Probe Name AssayProbe Sequence 5′ Primer 3′ Primer Neo GOA TGGGCACAAC GGTGGAGAGGAACACGGCGG AGACAATCGGC GCTATTCGGC CATCAG TG (SEQ ID (SEQ ID NO: 364)(SEQ ID NO: 362)  NO: 363) ULC-m1 GOA CCATTATGATG AGGTGAGGG TGACAAATGCCCCTCCATGCCTC TACAGATAAG TAATTATAGTGAT TCTGTTC TGTTATGAG CA(SEQ ID NO: 365) (SEQ ID (SEQ ID NO: 367) NO: 366) 1633h2 GOAATCAGCAGAAA GGGCAAGTC TGCAAACTGGAT (Vκ1-39Jκ5- CCAGGGAAAGC AGAGCATTAGGCAGCATAG specific) CCCT (SEQ ID CA (SEQ ID NO: 370) NO: 368)(SEQ ID NO: 369) mIgKd2 Retention GGCCACATTCC GCAAACAAAA CTGTTCCTCTAAAATGGGTTC ACCACTGGCC ACTGGACTCCAC (SEQ ID NO: 371) (SEQ ID AGTAAATGGAAANO: 372) (SEQ ID NO: 373) mIgKp15 Retention GGGCACTGGAT CACAGCTTGTAGAAGAAGCCTG ACGATGTATGG GCAGCCTCC TACTACAGCATC (SEQ ID NO: 374) (SEQ IDCGTTTTACAGTCA NO: 375) (SEQ ID NO: 376)

The NEO selection cassette introduced by the targeting constructs wasdeleted by transfecting ES cells with a plasmid that expresses FLP(FIGS. 23C and 23E). Optionally, the neomycin cassette may be removed bybreeding to mice that express FLP recombinase (e.g., U.S. Pat. No.6,774,279). Optionally, the neomycin cassette is retained in the mice.

Targeted ES cells described above were used as donor ES cells andintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method(see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) F0generation mice that are essentially fully derived from the donorgene-targeted ES cells allowing immediate phenotypic analyses NatureBiotech. 25(1):91-99. VELOCIMICE® independently bearing an engineeredhuman light chain gene that contains histidine residues mutated into oneor more positions along the sequence were made from the targeted EScells described above.

Pups were genotyped and pups heterozygous for the engineeredhistidine-modified human light chain were selected for characterizingexpression of the light chain and binding capabilities of the expressedantibodies. Primers and probes for genotyping of mice specificallycomprising a universal light chain gene with either three (H106/108/111;“1930”) or four (H105/105/108/111; “1927”) histidine modifications arelisted in Table 4 below and set forth in the Sequence Listing. Micecontaining histidine modification in their universal light chains arereferred herein as “HULC” mice (histidine universal light chain mice).

TABLE 4  Primers and Probes Used for Genotyping Probe Probe Name AssaySequence 5′ Primer 3′ Primer 1927jxn3 GOA 1927 (4 ACCATAGTCAAGCAGTCTGCAA CCCTTGGCCG His) mouse- CAGTACCCA CCTGAAGATTT AAGGTGATspecific (SEQ ID (SEQ ID (SEQ ID NO: 377) NO: 378) NO: 379) 1930jxn3GOA 1930(3 ATAGTCACAG AGTCTGCAACCT CCCTTGGCCG His) mouse- TACCCATCCGAAGATTTTGC AAGGTGAT specific (SEQ ID (SEQ ID (SEQ ID NO: 380) NO: 381)NO: 382)

Example 6.2 Analysis of Immune Response to Antigen in Mice withHistidine-Substituted Universal Light Chains

Cell surface receptor (“Antigen A”) was used as the immunogen toimmunize mice that were either heterozygous for expression of apre-arranged human kappa light chain utilizing Vκ1-39 and Jκ5 that has 4histidine substitutions in CDR3 (hereinafter “HULC 1927”) orheterozygous for expression of a pre-arranged human kappa light chainutilizing Vκ1-39 and Jκ5 that has 3 histidine substitutions in CDR3(hereinafter “HULC1930”), or homozygous WT mice. Pre-immune serum wascollected from the mice prior to the initiation of immunization. Theimmunogen was administered at 2.35 μg of protein for the initial primingimmunization mixed with 10 μg of CpG oligonucleotide as an adjuvant(Invivogen) in a volume of 25 μl via footpad (f.p.). Subsequently, micewere boosted via the same route with 2.35 μg of Antigen A along with 10μg of CpG and 25 μg of Adju-Phos (Brenntag) as adjuvants on days 3, 6,11, 13, 17, 20 for a total of 6 boosts. The mice were bled on days 15and 22 after the 4^(th) and 6^(th) boost, respectively. Their antiserumwas assayed for antibody titers to Antigen A.

Antibody serum titers against immunogen were determined by a standardELISA. To perform the ELISA, 96-well microtiter plates (ThermoScientific) were coated at 2 μg/ml with Antigen A in phosphate-bufferedsaline (PBS, Irvine Scientific) overnight at 4° C. The next day, plateswere washed with phosphate-buffered saline containing 0.05% Tween 20(PBS-T, Sigma-Aldrich) four times using a plate washer (MolecularDevices). Plates were then blocked with 250 μl of 0.5% bovine serumalbumin (BSA, Sigma-Aldrich) in PBS and incubated for 1 hour at roomtemperature. The plates were then washed four times with PBS-T. Serafrom immunized mice and pre-immune sera were serially diluted three-foldin 0.5% BSA-PBS starting at 1:300 or 1:1000, added to the blocked platesin duplicate, and then incubated for 1 hour at room temperature. Thelast two wells were left blank to be used as a secondary antibodycontrol (background control). The plates were again washed four timeswith PBS-T in a plate washer. Goat anti-mouse IgG-Fc-Horse RadishPeroxidase (HRP) conjugated secondary antibody (Jackson Immunoresearch)was then added to the plates at 1:5000/1:10,000 dilution and incubatedfor 1 hour at room temperature. Plates were then washed eight times withPBS-T and developed using TMB/H₂O₂ as substrate. The substrate wasincubated for 20 min and the reaction was stopped with 2 N sulfuric acid(H₂SO₄, VWR, cat#BDH3500-1) or 1 N phosphoric acid (JT Baker,Cat#7664-38-2). Plates were read on a spectrophotometer (Victor, PerkinElmer) at 450 nm. Antibody titers were computed using Graphpad PRISMsoftware.

The immune response induced in mice to the injected immunogen isrepresented as antibody titers, which is defined as the reciprocal ofthe highest serum dilution at which antigen binding absorbance istwo-fold higher over background. Therefore, the higher the number, thegreater the humoral immune response to the immunogen. Antibody titersinduced to the immunogen were very high in both strains of HULC mice andin the WT mice, with no significant differences observed among thestrains (FIG. 24).

Example 6.3 Generation of pH-Sensitive Monoclonal Antibodies

When a desired immune response to the immunogen was achieved in bothstrains of HULC mice and in the WT mice, splenocytes from each mousestrain were harvested and fused with mouse myeloma cells to generatehybridoma cells, which were allowed to grow in 96-well plates. After 10days of growth, supernatants from each hybridoma cell-containing wellwere screened via immunogen-specific ELISA to identify positive antigenbinding samples. For the ELISA, 96 well micro-titer plates were coatedwith 1 ug/mL of an anti-myc polyclonal antibody (Novus Biologicals,#NB600-34) overnight at 4° C. to immobilize the myc-tagged antigen,followed by blocking with a solution of 0.5% (w/v) BSA in PBS. Theplates were washed, the antigen solutions were added to the plates at aconcentration of 1 μg/mL and allowed to bind to the coated plate for 1hour at room temperature. Subsequently, supernatants from hybridomacells were added to the wells at 1:50 dilution and allowed to bind for 1hour at room temperature. The plate bound antibodies were detected usingan anti-mouse IgG polyclonal antibody conjugated with HRP (JacksonImmunoresearch, #115-035-164). TMB substrates were added to the plates(BD Biosciences, #51-2606KC/51-2607KC) and colorimetric signals weredeveloped according to manufacturer recommended protocol. The absorbancewas recorded at 450 nm on a Victor Wallac plate reader. Antigen positivesamples defined as having an OD equal to or greater than 0.5 (with thebaseline having OD of about 0.1) were subject to affinity screeningusing a real-time surface plasmon resonance biosensor (Biacore 4000).

Kinetic binding parameters (e.g., k_(a), k_(d), K_(D), t_(1/2), etc.)for antibody binding to the immunogen at neutral pH (pH 7.4) and atacidic pH (pH 6.0) were recorded. A Biacore CM4 sensor chip wasderivatized with a polyclonal goat anti-mouse Fc antibody to captureantibodies from the supernatant. A single concentration (100 nM) ofimmunogen was then injected over the antibody-captured surface at a flowrate of 30 μl/min. Antibody-antigen association was monitored for 1.5minutes and then the dissociation of antigen from the captured antibodywas monitored for 2.5 minutes. Kinetic association (k_(a)) anddissociation (k_(d)) rate constants were determined by processing andfitting the data to a 1:1 binding with a mass transport model usingBiacore 4000 Evaluation software version 1.0. Equilibrium dissociationconstants (K_(D)) and dissociative half-lives (t_(1/2)) were calculatedfrom the kinetic rate constants as: K_(D) (M)=k_(d)/k_(a); and t_(1/2)(min)=ln 2/(60*k_(d)). A set of samples that displayed decreased bindingat pH 6.0 as compared to that at pH 7.4 (pH sensitive) as well as a setof control samples that displayed no significant rate changes betweenthe pH 7.4 and pH 6.0 (pH insensitive controls) were selected to beproduced clonally. FIG. 25 depicts comparison of the number of totalantigen positives and the number of antigen positives displayingpH-sensitive antigen binding from HULC and WT mice.

Among the antigen positives, 18 and 7 clones isolated from twoheterozygous HULC1927 mice and two HULC1930 respectively, and 1 clonefrom the WT mouse, were made monoclonal. Supernatants of the monoclonalhybridomas were subject to neutral and low pH antigen dissociation rate(off-rate) analysis and cell pellets were used for light chain variabledomain DNA sequencing.

Example 6.4 Sequencing and Somatic Hypermutations in CDR3 Region ofVκ1-39Jκ5-Based Histidine Universal Light Chain Mice

Cell pellets from monoclonal hybridomas from HULC and WT mice were usedfor light chain variable domain DNA sequencing. From the 26 clones mademonoclonal (see Example 3.3 above) and subjected to sequencing, 15 wereconfirmed as using either a HULC or WT mouse light chain (MM and NN, seeTable 4). 14 clones were derived from HULC heterozygous mice (1927 or1930 mice) and 1 was derived from a WT mouse.

From the 14 antigen positive samples derived from HULC heterozygousmice, 12 of the monoclonal antibodies utilized their corresponding HULClight chain, while 2 utilized a WT mouse light chain. All but one of theHULC utilizing antibodies retained all of the introduced histidinemutations as shown in Table 3 (italicized antibody). Sequencing of cloneAA produced 2 different HULC sequences, which is reflected by twoentries in Table 5.

TABLE 5 Number of conserved histidine insertions and somatichypermutations in light sequences from clones utilizing the HULC lightchain Light Chain Sequences from mice utilizing HULC # Conserved His #Somatic # Somatic Clone Mouse Mutations Hypermutations HypermutationsName Strain in CDR3 in Framework in CDRs AA 1927 4 3 0 (Sequence 1) AA1927 4 1 1 (Sequence 2) BB 1927 4 3 3 CC 1927 4 0 0 DD 1927 3 1 1 EE1927 4 2 2 FF 1927 4 0 1 GG 1927 4 1 1 HH 1927 4 2 0 II 1930 3 1 1 JJ1930 3 4 5 KK 1930 3 1 2 LL 1930 3 1 0

Example 6.5 pH-Dependent Binding of Monoclonal Antibodies Generated inVκ1-39Jκ5-Based Histidine Universal Light Chain Mice

In order to further assess the pH-dependent binding characteristics ofthe monoclonal antibodies isolated from HULC and WT mice, bindingexperiments were carried out in which the antibody/antigen associationphase was observed at neutral pH and the antibody/antigen dissociationphase was observed at either neutral or acidic pHs.

A Biacore CM4 sensor chip was derivatized with a polyclonal rabbitanti-mouse Fc antibody. Monoclonal antibody supernatants were capturedonto the anti-mouse Fc sensor surface. Two concentrations, 50 nM (induplicate) and 16.7 nM, of the immunogen were injected over themonoclonal antibody captured surface at a flow rate of 30 μl/min.Antibody-antigen association was monitored at pH 7.4 for 4 minutes andthen the dissociation of antigen from the captured monoclonal antibodywas monitored for 15 minutes at either pH 7.4 or 6.0. Dissociation(k_(d)) rate constants were determined by processing and fitting thedata using Scrubber version 2.0 curve fitting software and are shown inTable 6. Dissociative half-lives (t_(1/2)) were calculated from thedissociation rate constants as: t_(1/2) (min)=(ln 2/k_(d))/60, and areshown in Table 6. Sensorgrams depicting the association/dissociationcharacteristics of several antibodies listed in Table 4 under thevarious pH conditions are shown graphically in FIG. 26. The individuallines in each graph represent the binding responses at differentconcentrations of the respective antibodies. All experiments werecarried out at 25° C. Dissociative half-life values (t½) are noted abovethe respective sensorgrams. Response is measured in RU.

TABLE 6 Dissociation (k_(d)) rate constants and dissociative half-lives(t_(1/2)) of monoclonal HULC or WT antibodies binding to their immunogenat neutral and low pH. pH 7.4 Association/pH 7.4 pH 7.4 Association/pH6.0 Dissociation Dissociation 50 nM 50 nM Light neutral immunogen lowimmunogen pH 6.0/pH 7.4 Clone chain mAb bound k_(d) t½ mab bound k_(d)t½ ratio Name used capture (RU) (1/s) (min) capture (RU) (1/s) (min)k_(d) t½ AA HULC 129 70 5.60E−05 206 122 73 2.18E−04 53 3.9 0.3 (1927)BB HULC 350 165 6.00E−04 19 378 185 2.20E−03 5 3.7 0.3 (1927) CC HULC611 251 2.03E−04 57 545 226 6.68E−03 2 33.0 0.03 (1927) DD HULC 182 753.55E−04 33 168 74 6.44E−04 18 1.8 0.6 (1927) HH HULC 268 92 1.36E−04 85251 91 5.39E−04 21 4.0 0.3 (1927) GG HULC 353 110 2.78E−04 42 328 1028.97E−04 13 3.2 0.3 (1927) FF HULC 334 202 4.79E−05 241 364 220 6.90E−05167 1.4 0.7 (1927) EE HULC 339 124 5.08E−04 23 299 120 4.66E−04 25 0.91.1 (1927) II HULC 387 174 1.22E−04 95 334 147 2.14E−04 54 1.8 0.6(1930) JJ HULC 363 14 9.83E−04 12 333 12 5.30E−04 22 0.5 1.9 (1930) KKHULC 490 303 7.41E−05 156 484 295 1.29E−04 90 1.7 0.6 (1930) LL HULC 63641 3.09E−04 37 597 36 5.77E−04 20 1.9 0.5 (1930) MM* WT 245 6 NA NA 2036 NA NA NA NA (from 1927 mouse) NN WT 394 231 5.26E−04 22 378 2319.35E−04 12 1.8 0.6 (from 1927 mouse) OO WT 413 89 2.94E−04 39 400 833.57E−04 32 1.2 0.8 *k_(d) and t_(1/2) values could not be determineddue to low antigen binding signal

Example 7 Engineering of Genetically Modified Mouse Comprising aHistidine-Substituted Vκ3-20Jκ1 Universal Light Chain

A mouse comprising a common Vκ3-20Jκ1 light chain was generated asdescribed in, e.g., U.S. patent application Ser. Nos. 13/022,759,13/093,156, 13/412,936, and 13/488,628 (Publication Nos. 2011/0195454,2012/0021409, 2012/0192300, and 2013/0045492, respectively), and inExample 1 above. The amino acid sequence of the germline universalVκ3-20R1 light chain variable domain is set forth in SEQ ID NO:383.

Histidine substitutions were introduced into the Vκ3-20R1 universallight chain targeting vector and mice generated from the same using asimilar strategy to the one described above in Example 3 for Vκ1-39Jκ5histidine modified universal light chain mice (HULC 1927 and 1930).

Briefly, the strategy for generating a histidine-modified Vκ3-20R1universal light chain targeting vector is summarized in FIGS. 29A-129D.A plasmid used for generating a targeting vector for common (universal)light chain mouse (“ULC mouse,” described in, e.g., US2011/0195454A1),containing pBS+FRT-Ub-Hyg-FRT+mouse Vκ3-7 leader+human Vκ3-20Jκ1 wasmodified by site directed mutagenesis (QuickChange Lightning Kit) toreplace Q105, Q106, Y107 and S109 or Q105, Q106 and S109 (see alignmentin FIG. 27) with histidine residues in the CDR3 region usingsite-directed mutagenesis primers shown in FIG. 28 (See FIG. 29A forthis engineering step). Resultant vectors (H105/106/107/109 andH105/106/109) were modified further and ligated into a vector comprisingmouse Igκ constant region, mouse enhancers, a mouse 3′ homology arm anda SPEC cassette (FIG. 29B). Further modification involved ligation intoa vector carrying 5′ mouse arm and comprising Frt-UB-NEO-Frt cassette(FIG. 29B). Resultant targeting vectors were electroporated into EScells comprising deletion of the mouse Igκ variable locus (comprising κvariable and joining gene segments) (FIGS. 29C-29D).

Positive ES cell clones were confirmed by using a modification of alleleassay (Valenzuela et al.) using probes specific for the engineeredVκ3-20κJ1 light chain region inserted into the endogenous κ light chainlocus. Primers and probes used in the assay are shown in Table 7 belowand set forth in the Sequence Listing; the locations of the probes aredepicted in FIGS. 29C-29D.

TABLE 7  Primers and Probes Used for ES Cell Screening Probe Probe NameAssay Sequence 5′ Primer 3′ Primer Neo GOA TGGGCACAAC GGTGGAGAGGAACACGGC AGACAATCGG GCTATTCGGC GGCATCAG CTG (SEQ ID (SEQ ID (SEQ IDNO: 363) NO: 364) NO: 362) ULC-m1 GOA CCATTATGATG AGGTGAGGG TGACAAATGCCTCCATGCCT TACAGATAAG CCTAATTATA CTCTGTTC TGTTATGAG GTGATCA (SEQ ID(SEQ ID (SEQ ID NO: 365) NO: 366) NO: 367) 1635h2 GOA AAAGAGCCACTCCAGGCACC AAGTAGCTGC (Vκ3-20Jκ1 CCTCTCCTGC CTGTCTTTG TGCTAACACTspecific) AGGG CTGACT (SEQ ID (SEQ ID (SEQ ID NO: 391) NO: 389) NO: 390)mIgKd2 Reten- GGCCACATTC GCAAACAAAA CTGTTCCTCT tion CATGGGTTC ACCACTGGCCAAAACTGGAC (SEQ ID (SEQ ID TCCACAGTAA NO: 371) NO: 372) ATGGAAA (SEQ IDNO: 373) mIgKp15 Reten- GGGCACTGGA CACAGCTTGT AGAAGAAGCC tionTACGATGTATG GCAGCCTCC TGTACTACAG G (SEQ ID CATCCGTTTT (SEQ ID NO: 375)ACAGTCA NO: 374) (SEQ ID NO: 376)

The NEO selection cassette introduced by the targeting constructs isdeleted by transfecting ES cells with a plasmid that expresses FLP(FIGS. 29C and 29D). Optionally, the neomycin cassette may be removed bybreeding to mice that express FLP recombinase (e.g., U.S. Pat. No.6,774,279). Optionally, the neomycin cassette is retained in the mice.

Targeted ES cells described above are used as donor ES cells andintroduced into an 8-cell stage mouse embryo by the VELOCIMOUSE® method(see, e.g., U.S. Pat. No. 7,294,754 and Poueymirou et al. (2007) F0generation mice that are essentially fully derived from the donorgene-targeted ES cells allowing immediate phenotypic analyses NatureBiotech. 25(1):91-99). VELOCIMICE® independently bearing an engineeredhuman light chain gene that contains histidine residues mutated into oneor more positions along the sequence are made from the targeted ES cellsdescribed above.

Pups are genotyped and pups heterozygous for the engineeredhistidine-modified human light chain are selected for characterizingexpression of the light chain and binding capabilities of the expressedantibodies. Primers and probes for genotyping of mice specificallycomprising a universal light chain gene with either three (H105/106/109;“6183”) or four (H105/105/108/111; “6181”) histidine modifications arelisted in Table 6 below and set forth in the Sequence Listing. Micecontaining histidine modification in their universal light chains arereferred herein as “HULC” mice (histidine universal light chain mice).

TABLE 8  Primers and Probes Used for Genotyping Probe Probe Name AssaySequence 5′ Primer 3′ Primer hVI494-1 GOA 6181 CTGTCATCACC GCAGACTGGACCGAACGTCCAA (4 His) ATGG GCCTGAAGAT GGTGAGTG mouse- (SEQ ID TTT (SEQ IDspecific NO: 392) (SEQ ID NO: 394) NO: 393) hVI495-1 GOA 6183TACTGTCATCA  GCAGACTGGA CCGAACGTCCAA (3 His) CTATGG GCCTGAAGAT GGTGAGTGmouse- (SEQ ID TT (SEQ ID specific NO: 395) (SEQ ID NO: 397) NO: 396)

Mice are immunized with antigen of interest and tested for ability togenerate antibodies with pH-dependent binding.

Example 8 Breeding of Mice Comprising a Histidine-Substituted SingleRearranged Human Universal Light Chain Mouse (HULC)

This Example describes several other genetically modified mouse strainsthat can be bred to any one of the HULC mice described herein to createmultiple genetically modified mouse strains harboring multiplegenetically modified immunoglobulin loci.

Endogenous Igλ Knockout (KO).

To optimize the usage of the engineered light chain locus, any one ofthe HULC animals described above (e.g., comprising Vκ1-39Jκ5 orVκ3-20Jκ1 histidine-substituted universal light chain) may be bred toanother mouse containing a deletion in the endogenous λ light chainlocus. In this manner, the progeny obtained will express, as their onlylight chain, the rearranged histidine-substituted human germline lightchain region as described in Examples 3 and 4 above. Breeding isperformed by standard techniques recognized in the art and,alternatively, by a commercial breeder (e.g., The Jackson Laboratory).Mouse strains bearing an engineered histidine-substituted light chainlocus and a deletion of the endogenous λ light chain locus are screenedfor presence of the unique light chain region and absence of endogenousmouse λ light chains.

Humanized Endogenous Heavy Chain Locus. Mice bearing an engineered humangermline light chain locus (HULC mice) are bred with mice that contain areplacement of the endogenous mouse heavy chain variable gene locus withthe human heavy chain variable gene locus (see U.S. Pat. No. 6,596,541;the VELOCIMMUNE® mouse, Regeneron Pharmaceuticals, Inc.). TheVELOCIMMUNE® mouse comprises a genome comprising human heavy chainvariable regions operably linked to endogenous mouse constant regionloci such that the mouse produces antibodies comprising a human heavychain variable domain and a mouse heavy chain constant region inresponse to antigenic stimulation.

Mice bearing a replacement of the endogenous mouse heavy chain variableregion locus with the human heavy chain variable region locus and ahistidine-substituted single rearranged human light chain variableregion at the endogenous κ light chain locus are obtained. Reversechimeric antibodies containing somatically mutated heavy chains (humanheavy chain variable domain and mouse C_(H)) with ahistidine-substituted single human light chain (HULC, human light chainvariable domain and mouse C_(L)) are obtained upon immunization with anantigen of interest. pH-dependent human antibodies generated in suchmice are identified using antibody isolation and screening methods knownin the art or described above. Variable light and heavy chain regionnucleotide sequences of B cells expressing the antibodies, e.g.,pH-sensitive antibodies, are identified, and fully human antibodies aremade by fusion of the variable heavy and light chain region nucleotidesequences to human C_(H) and C_(L) nucleotide sequences, respectively,in a suitable expression system.

Example 9 Progeny of Genetically Modified Mice

Mice bearing an engineered human germline light chain locus comprising alimited repertoire of rearranged human light chain variable regionsequences or a single rearranged human light chain variable regionsequence (HULC mice) described herein are bred with mice that contain ahistidine-modified human heavy chain variable gene locus describedherein. Mice are obtained, and the presence of a light chain sequencecontaining histidine-modified human light chain variable region and aheavy chain sequence containing a histidine-modified human heavy chainvariable region is confirmed by genotyping.

Reverse chimeric antibodies containing histidine-modified heavy chains(human histidine-modified heavy chain variable domain and mouse C_(H))and histidine-modified single human light chain (HULC, humanhistidine-modified light chain variable domain and mouse C_(L)) areobtained upon immunization with an antigen of interest. pH-dependenthuman antibodies generated in such mice are identified using antibodyisolation and screening methods known in the art or described above.Variable light and heavy chain region nucleotide sequences of B cellsexpressing the antibodies, e.g., pH-sensitive antibodies, areidentified, and fully human antibodies are made by fusion of thevariable heavy and light chain region nucleotide sequences to humanC_(H) and C_(L) nucleotide sequences, respectively, in a suitableexpression system.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims. While the describedinvention has been described with reference to the specific embodimentsthereof it should be understood by those skilled in the art that variouschanges may be made and equivalents may be substituted without departingfrom the true spirit and scope of the invention. In addition, manymodifications may be made to adopt a particular situation, material,composition of matter, process, process step or steps, to the objectivespirit and scope of the described invention. All such modifications areintended to be within the scope of the claims appended hereto.

Entire contents of all non-patent documents, patent applications andpatents cited throughout this application are incorporated by referenceherein in their entirety. U.S. patent application Ser. Nos. 13/832,309and 13/832,247, each filed 15 Mar. 2013, are hereby incorporated byreference.

What is claimed is:
 1. A method for making a non-human animal that makesantibody variable domains with a histidine amino acid encoded by ahistidine codon substituted or inserted in the germline genome,comprising: modifying the non-human animal in its germline to compriseeither or both (i) at an endogenous non-human immunoglobulin heavy chainlocus an unrearranged immunoglobulin heavy chain variable region genesequence comprising in a complementary determining region 3 (CDR3)encoding sequence a substitution of at least one non-histidine codonwith a histidine codon or an insertion of at least one histidine codon,and (ii) at an endogenous non-human immunoglobulin light chain locus anunrearranged immunoglobulin light chain variable region gene sequencecomprising in a CDR3 encoding sequence a substitution of at least onenon-histidine codon with a histidine codon or an insertion of at leastone histidine codon; such that the non-human animal generates in vivo adiverse repertoire of antibodies, each of which is specific for anantigen of interest and retains at least one histidine amino acidencoded by the substituted or inserted histidine codon, wherein the atleast one histidine amino acid is present in a CDR3 of a heavy chainvariable domain and/or a CDR3 of a light chain variable domain.
 2. Themethod of claim 1, wherein the unrearranged immunoglobulin heavy chainvariable gene sequence is an unrearranged human immunoglobulin heavychain variable region nucleotide sequence comprising at least oneunrearranged human V_(H) gene segment, at least one unrearranged humanD_(H) gene segment, and at least one unrearranged human J_(H) genesegment, and wherein the at least one unrearranged human D_(H) genesegment comprises the substitution of at least one non-histidine codonwith a histidine codon or the insertion of at least one histidine codon.3. The method of claim 1, wherein the unrearranged immunoglobulin lightchain gene sequence is an unrearranged human immunoglobulin light chainvariable region nucleotide sequence comprising at least one unrearrangedhuman V_(L) gene segment and at least one unrearranged human J_(L) genesegment, and wherein the at least one unrearranged human V_(L) genesegment comprises in a CDR3 encoding sequence the substitution of atleast one non-histidine codon with a histidine codon or the insertion ofat least one histidine codon.
 4. The method of claim 1, wherein thenon-human animal is a rodent.
 5. The method of claim 4, wherein therodent is selected from a mouse, a rat, and a hamster.
 6. A method formaking a non-human animal that makes antibody variable domains with ahistidine amino acid encoded by a histidine codon substituted orinserted in the germline genome, comprising: modifying the non-humananimal in its germline to comprise (i) at an endogenous non-humanimmunoglobulin heavy chain locus an unrearranged immunoglobulin heavychain variable region gene sequence comprising in a CDR3 encodingsequence a substitution of at least one non-histidine codon with ahistidine codon or an insertion of at least one histidine codon, and(ii) at an endogenous non-human immunoglobulin light chain locus arearranged immunoglobulin light chain variable region gene sequencecomprising in a CDR3 encoding sequence a substitution of at least onenon-histidine codon with a histidine codon or an insertion of at leastone histidine codon; such that the non-human animal generates in vivo adiverse repertoire of antibodies, each of which is specific for anantigen of interest and retains at least one histidine amino acidencoded by the substituted or inserted histidine codon, wherein the atleast one histidine amino acid is present in a CDR3 of a light chainvariable domain.
 7. The method of claim 6, wherein the non-human animalis a rodent.
 8. The method of claim 7, wherein the rodent is selectedfrom a mouse, a rat, and a hamster.
 9. The method of claim 1, whereinthe modifying step comprises replacing at the endogenous non-humanimmunoglobulin heavy chain locus an endogenous non-human immunoglobulinheavy chain variable region gene with an unrearranged humanimmunoglobulin heavy chain variable region gene sequence comprising atleast one unrearranged human V_(H) gene segment, at least oneunrearranged human D_(H) gene segment, and at least one unrearrangedhuman J_(H) gene segment, and in a CDR3 encoding sequence thesubstitution of at least one non-histidine codon with a histidine codonor the insertion of at least one histidine codon, and/or replacing atthe endogenous non-human immunoglobulin light chain locus an endogenousnon-human immunoglobulin light chain variable region gene sequence withan unrearranged human immunoglobulin light chain variable region genesequence comprising at least one unrearranged human V_(L) gene segmentand unrearranged human J_(L) gene segment, and in a CDR3 encodingsequence the substitution of at least one non-histidine codon with ahistidine codon or the insertion of at least one histidine codon. 10.The method of claim 1, wherein the modifying step comprises replacing atthe endogenous non-human immunoglobulin heavy chain locus an endogenousnon-human immunoglobulin heavy chain variable region gene sequence in afirst non-human animal with an unrearranged human immunoglobulin heavychain variable region gene sequence comprising at least one unrearrangedhuman V_(H) gene segment, at least one unrearranged human D_(H) genesegment, and at least one unrearranged human J_(H) gene segment, and ina CDR3 encoding sequence the substitution of at least one non-histidinecodon with a histidine codon or the insertion of at least one histidinecodon, replacing at the endogenous non-human immunoglobulin light chainlocus an endogenous non-human immunoglobulin light chain variable regiongene sequence in a second non-human animal with an unrearranged humanimmunoglobulin light chain variable region gene sequence comprising atleast one unrearranged human V_(L) gene segment and at least oneunrearranged human J_(L) gene segment, and in a CDR3 encoding sequencethe substitution of at least one non-histidine codon with a histidinecodon or the insertion of at least one histidine codon, and breeding thefirst and second non-human animals to generate a third non-human animalcomprising a replacement of the endogenous non-human immunoglobulinheavy chain variable region gene sequence with the unrearranged humanimmunoglobulin heavy chain variable region gene sequence and areplacement of the endogenous immunoglobulin light chain variable regiongene sequence with the unrearranged human light chain variable regiongene sequence, wherein the first, second and third non-human animals arethe same species.
 11. The method of claim 1, wherein the unrearrangedimmunoglobulin heavy chain variable region gene sequence and/or theunrearranged immunoglobulin light chain variable region gene sequencefurther comprises an additional substituted or inserted histidine codonin a complementary determining region 1 (CDR1) encoding sequence, acomplementary determining region 2 (CDR2) encoding sequence anN-terminal encoding sequence or a loop encoding sequence.
 12. The methodof claim 2, wherein the unrearranged immunoglobulin heavy chain variablegene sequence is operably linked to an endogenous non-humanimmunoglobulin heavy chain constant region gene sequence and/or theunrearranged immunoglobulin light chain variable gene sequence isoperably linked to an endogenous non-human immunoglobulin light chainconstant region gene sequence.
 13. The method of claim 6, wherein themodifying step comprises replacing at the endogenous non-humanimmunoglobulin heavy chain locus an endogenous non-human immunoglobulinheavy chain variable region gene with an unrearranged humanimmunoglobulin heavy chain variable region gene sequence comprising atleast one unrearranged human V_(H) gene segment, at least oneunrearranged human D_(H) gene segment, and at least one unrearrangedhuman J_(H) gene segment, and in a CDR3 encoding sequence thesubstitution of at least one non-histidine codon with a histidine codonor the insertion of at least one histidine codon; and replacing at theendogenous non-human immunoglobulin light chain locus an endogenousnon-human immunoglobulin light chain variable region gene sequence witha rearranged human immunoglobulin light chain variable region genesequence comprising in a CDR3 encoding sequence the substitution of atleast one non-histidine codon with a histidine codon or the insertion ofat least one histidine codon.
 14. The method of claim 6, wherein themodifying step comprises replacing at the endogenous non-humanimmunoglobulin heavy chain locus an endogenous non-human immunoglobulinheavy chain variable region gene sequence in a first animal with anunrearranged human immunoglobulin heavy chain variable region genesequence comprising unrearranged human V_(H), unrearranged human D_(H),and unrearranged human J_(H) gene segments, and in a CDR3 encodingsequence the substitution of at least one non-histidine codon with ahistidine codon or an insertion of at least one histidine codon,replacing at the endogenous non-human immunoglobulin light chain locusan endogenous non-human immunoglobulin light chain variable region genesequence in a second animal with a rearranged human immunoglobulin lightchain variable region gene sequence comprising in a CDR3 encodingsequence the substitution of at least one non-histidine codon with ahistidine codon or an insertion of at least one histidine codon, andbreeding the first and second non-human animals to generate a thirdnon-human animal comprising a replacement of the endogenous non-humanimmunoglobulin heavy chain variable region gene sequence with theunrearranged human immunoglobulin heavy chain variable region genesequence and a replacement of the endogenous immunoglobulin light chainvariable region gene sequence with the rearranged human light chainvariable region gene sequence, wherein the first, second and thirdnon-human animals are the same species.
 15. The method of claim 6,wherein the unrearranged immunoglobulin heavy chain variable region genesequence and/or the rearranged immunoglobulin light chain variableregion gene sequence further comprises an additional substituted orinserted histidine codon in a CDR1 encoding sequence, a CDR2 encodingsequence, an N-terminal encoding sequence or a loop encoding sequence.16. The method of claim 6, wherein the rearranged immunoglobulin lightchain variable region gene sequence is derived from a human Vκ1-39 orVκ3-20 gene segment.
 17. The method of claim of claim 16, wherein therearranged immunoglobulin light chain variable region is derived from arearranged human Vκ1-39/Jκ5 or Vκ3-20/Jκ1 gene sequence.
 18. The methodof claim 6, wherein the unrearranged immunoglobulin heavy chain variablegene sequence is operably linked to an endogenous non-humanimmunoglobulin heavy chain constant region gene sequence.
 19. The methodof claim 3, wherein the unrearranged human immunoglobulin light chainvariable region light chain gene sequence comprises no more than twohuman V_(L) gene segments.
 20. The method of claim 19, wherein each ofthe no more than two human V_(L) gene segments comprises in a CDR3encoding sequence a substitution of at least one non-histidine codonwith a histidine codon or an insertion of at least one histidine codon.